Activity of Fe—S cluster requiring proteins

ABSTRACT

The present invention is related to a recombinant host cell, in particular a yeast cell, comprising a dihydroxy-acid dehydratase polypeptide. The invention is also related to a recombinant host cell having increased specific activity of the dihydroxy-acid dehydratase polypeptide as a result of increased expression of the polypeptide, modulation of the Fe—S cluster biosynthesis of the cell, or a combination thereof. The present invention also includes methods of using the host cells, as well as, methods for identifying polypeptides that increase the flux in an Fe—S cluster biosynthesis pathway in a host cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 13/837,921, filed on Mar. 15, 2013 and U.S. patentapplication Ser. No. 13/837,893, filed on Mar. 15, 2013 both of whichare continuations of and claim priority to U.S. patent application Ser.No. 13/029,558, filed on Feb. 17, 2011, now U.S. Pat. No. 9,297,016,issued on Mar. 29, 2016, which claims the benefit of U.S. ProvisionalAppl. No. 61/305,333, filed Feb. 17, 2010, each of which is incorporatedby reference in its entirety.

SEQUENCE LISTING INFORMATION

The content of the electronically submitted sequence listing in ASCIItext file (Name: 20161031_CL4842USCNT3_ST25) filed with the applicationis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates generally to the fields of microbiology andbiochemistry. Specifically, the present invention is related to arecombinant host cell, in particular a yeast cell, comprising adihydroxy-acid dehydratase polypeptide. The invention is also related toa recombinant host cell having increased specific activity of thedihydroxy-acid dehydratase polypeptide as a result of increasedexpression of the polypeptide, modulation of the Fe—S clusterbiosynthesis activity of the cell, or a combination thereof. The presentinvention also includes methods of using the host cells, as well asmethods for identifying polypeptides that increase the flux in an Fe—Scluster biosynthesis pathway in a host cell.

Background of the Invention

Iron-sulfur (Fe—S) clusters serve as cofactors or prosthetic groupsessential for the normal function of the class of proteins that containthem. In the class of Fe—S cluster containing proteins, the Fe—Sclusters have been found to play several roles. When proteins of thisclass are first synthesized by the cell, they lack the Fe—S clustersrequired for their proper function and are referred to as apoproteins.Fe—S clusters are made in a series of reactions by proteins involved inFe—S cluster biosynthesis and are transferred to the apo-proteins toform the functional Fe—S cluster containing holoproteins.

One such protein that requires Fe—S clusters for proper function isdihydroxy-acid dehydratase (DHAD) (E.C. 4.2.1.9). DHAD catalyzes theconversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, and of2,3-dihydroxymethylvalerate to α-ketomethylvalerate. The DHAD enzyme ispart of naturally occurring biosynthetic pathways producing the branchedchain amino acids, (i.e., valine, isoleucine, leucine), and pantothenicacid (vitamin B5). DHAD catalyzed conversion of 2,3-dihydroxyisovalerateto α-ketoisovalerate is also a common step in the multiple isobutanolbiosynthetic pathways that are disclosed in U.S. Patent Appl. Pub. No.US 20070092957 A1, incorporated by reference herein. Disclosed thereinis, e.g., the engineering of recombinant microorganisms for theproduction of isobutanol.

High levels of DHAD activity are desired for increased production ofproducts from biosynthetic pathways that include this enzyme activity,including, e.g., enhanced microbial production of branched chain aminoacids, pantothenic acid, and isobutanol. Isobutanol, in particular, isuseful as a fuel additive, and its ready availability may reduce thedemand for petrochemical fuels. However, since all known DHAD enzymesrequire a Fe—S cluster for their function, they must be expressed in ahost having the genetic machinery to provide the Fe—S clusters requiredby these proteins. In yeast, mitochondria play an essential role in Fe—Scluster biosynthesis. If the DHAD is to be functionally expressed inyeast cytosol, a system to transport the requisite Fe—S precursor orsignal from mitochondria and assemble the Fe—S cluster on the cytosolicapoprotein is required. Prior to the work of the present inventors, itwas previously unknown whether yeast could provide Fe—S clusters for anyDHAD located in the cytoplasm (since native yeast DHAD is located in themitochondria) and more importantly when the DHAD is expressed at highlevels in the cytoplasm.

Under certain conditions the rate of synthesis of Fe—S cluster requiringapo-proteins may exceed the cell's ability to synthesize and assembleFe—S clusters for them. Cluster-less apo-proteins that accumulate underthese conditions cannot carry out their normal function. Such conditionscan include 1) the expression of a heterologous Fe—S cluster requiringprotein especially in high amounts, 2) the expression of a native Fe—Scluster biosynthesis protein at higher levels than normal, or 3) a statewhere the host cell's ability to synthesize Fe—S clusters isdebilitated.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is the surprising discovery that recombinant host cellsexpressing a high level of a heterologous Fe—S cluster requiring proteincan supply the complement of Fe—S clusters for that protein if thelevel(s) of at least one Fe uptake, utilization, and/or Fe—S clusterbiosynthesis protein are altered.

Provided herein are recombinant host cells comprising at least oneheterologous polynucleotide encoding a polypeptide having dihydroxy-aciddehydratase activity wherein said at least one heterologouspolynucleotide comprises a high copy number plasmid or a plasmid with acopy number that can be regulated. Also provided are recombinant hostcells comprising at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity wherein said atleast one heterologous polynucleotide is integrated at least once in therecombinant host cell DNA. Also provided are recombinant host cellscomprising at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity, wherein saidhost cell comprises at least one deletion, mutation, and/or substitutionin an endogenous gene encoding a polypeptide affecting iron metabolismor Fe—S cluster biosynthesis. Also provided are recombinant host cellscomprising at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity and at least oneheterologous polynucleotide encoding a polypeptide affecting ironmetabolism or Fe—S cluster biosynthesis.

In embodiments, said heterologous polynucleotide encoding a polypeptideaffecting Fe—S cluster biosynthesis is selected from the groupconsisting of the genes in Tables 7, 8 and 9. In embodiments, saidheterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis is selected from the group consisting of AFT1,AFT2, CCC1, FRA2, and GRX3, and combinations thereof. In embodiments,polypeptide is encoded by a polynucleotide that is constitutive mutant.In embodiments, said constitutive mutant is selected from the groupconsisting of AFT1 L99A, AFT1 L102A, AFT1 C291F, AFT1 C293F, andcombinations thereof. In embodiments said polypeptide affecting Fe—Scluster biosynthesis is encoded by a polynucleotide comprising a highcopy number plasmid or a plasmid with a copy number that can beregulated. In embodiments, said polypeptide affecting Fe—S clusterbiosynthesis is encoded by a polynucleotide integrated at least once inthe recombinant host cell DNA. In embodiments, the at least onedeletion, mutation, and/or substitution in an endogenous gene encoding apolypeptide affecting Fe—S cluster biosynthesis is selected from thegroup consisting of CCC1, FRA2, and GRX3, and combinations thereof. Inembodiments, the at least one heterologous polynucleotide encoding apolypeptide affecting Fe—S cluster biosynthesis is selected from thegroup consisting of AFT1, AFT2, their mutants, and combinations thereof.

In embodiments, said at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity is expressed inmultiple copies. In embodiments, said at least one heterologouspolynucleotide comprises a high copy number plasmid or a plasmid with acopy number that can be regulated. In embodiments, said at least oneheterologous polynucleotide is integrated at least once in therecombinant host cell DNA. In embodiments, said Fe—S clusterbiosynthesis is increased compared to a recombinant host cell havingendogenous Fe—S cluster biosynthesis.

In embodiments, said host cell is a yeast host cell. In embodiments,said yeast host cell is selected from the group consisting ofSaccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces,Yarrowia, Issatchenkia and Pichia.

In embodiments, said heterologous polypeptide having dihydroxy-aciddehydratase activity is expressed in the cytosol of the host cell. Inembodiments, said heterologous polypeptide having dihydroxy-aciddehydratase activity has an amino acid sequence that matches the ProfileHMM of Table 12 with an E value of <10⁻⁵ wherein the polypeptide furthercomprises all three conserved cysteines, corresponding to positions 56,129, and 201 in the amino acids sequences of the Streptococcus mutansDHAD enzyme corresponding to SEQ ID NO:168. In embodiments, saidheterologous polypeptide having dihydroxy-acid dehydratase activity hasan amino acid sequence with at least about 90% identity to SEQ ID NO:168 or SEQ ID NO: 232. In embodiments said polypeptide havingdihydroxy-acid dehydratase activity has a specific activity selectedfrom the group consisting of: greater than about 5-fold with respect tothe control host cell comprising at least one heterologouspolynucleotide encoding a polypeptide having dihydroxy-acid dehydrataseactivity, greater than about 8-fold with respect to the control hostcell comprising at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity, or greater thanabout 10-fold with respect to the control host cell comprising at leastone heterologous polynucleotide encoding a polypeptide havingdihydroxy-acid dehydratase activity. In embodiments said polypeptidehaving dihydroxy-acid dehydratase activity has a specific activityselected from the group consisting of: greater than about 3-fold withrespect to a control host cell comprising at least one heterologouspolynucleotide encoding a polypeptide having dihydroxy-acid dehydrataseactivity and greater than about 6-fold with respect to the control hostcell comprising at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity. In embodiments,said polypeptide having dihydroxy-acid dehydratase activity has aspecific activity selected from the group consisting of: greater thanabout 0.25 U/mg; greater than about 0.3 U/mg; greater than about 0.5U/mg; greater than about 1.0 U/mg; greater than about 1.5 U/mg; greaterthan about 2.0 U/mg; greater than about 3.0 U/mg; greater than about 4.0U/mg; greater than about 5.0 U/mg; greater than about 6.0 U/mg; greaterthan about 7.0 U/mg; greater than about 8.0 U/mg; greater than about 9.0U/mg; greater than about 10.0 U/mg; greater than about 20.0 U/mg; andgreater than about 50.0 U/mg.

In embodiments said recombinant host cell produces isobutanol, and inembodiments, said recombinant host cell comprises an isobutanolbiosynthetic pathway.

Also provided herein are methods of making a product comprising:providing a recombinant host cell; and contacting the recombinant hostcell of with a fermentable carbon substrate in a fermentation mediumunder conditions wherein said product is produced, wherein the productis selected from the group consisting of branched chain amino acids,pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol,and combinations thereof. In embodiments, the methods further compriseoptionally recovering said product. In embodiments, the methods furthercomprise recovering said product.

Also provided are methods of making isobutanol comprising: providing arecombinant host cell; contacting the recombinant host cell with afermentable carbon substrate in a fermentation medium under conditionswherein isobutanol is produced. In embodiments, the methods furthercomprise optionally recovering said isobutanol. In embodiments, themethods further comprise recovering said isobutanol.

Also provided are methods for the conversion of 2,3-dihydroxyisovalerateto α-ketoisovalerate comprising: providing a recombinant host cell;growing the recombinant host cell of under conditions where the2,3-dihydroxyisovalerate is converted to α-ketoisovalerate. Inembodiments, the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate compared to a control host cell comprising at leastone heterologous polynucleotide encoding a polypeptide havingdihydroxy-acid dehydratase activity is increased in an amount selectedfrom the group consisting of: (a) at least about 5%; (b) at least about10%; (c) at least about 15%; (d) at least about 20%; (e) at least about25%; (f) at least about 30%; (g) at least about 35%; (h) at least about40%; (i) at least about 45%; (j) at least about 50%; (k) at least about60%; (l) at least about 70%; (m) at least about 80%; (n) at least about90%; and (o) at least about 95%.

Also provided are methods for increasing the specific activity of aheterologous polypeptide having dihydroxy-acid dehydratase activity in arecombinant host cell comprising: providing a recombinant host cell; andgrowing the recombinant host cell of under conditions whereby theheterologous polypeptide having dihydroxy-acid dehydratase activity isexpressed in functional form having a specific activity greater than thesame host cell lacking said heterologous polypeptide.

Also provided are methods for increasing the flux in an Fe—S clusterbiosynthesis pathway in a host cell comprising: providing a recombinanthost cell; and growing the recombinant host cell under conditionswhereby the flux in the Fe—S cluster biosynthesis pathway in the hostcell is increased.

Also provide are methods of increasing the activity of an Fe—S clusterrequiring protein in a recombinant host cell comprising: providing arecombinant host cell comprising an Fe—S cluster requiring protein;changing the expression or activity of a polypeptide affecting Fe—Scluster biosynthesis in said host cell; and growing the recombinant hostcell under conditions whereby the activity of the Fe—S cluster requiringprotein is increased. In embodiments, said increase in activity is anamount selected from the group consisting of: greater than about 10%;greater than about 20%; greater than about 30%; greater than about 40%;greater than about 50%; greater than about 60%; greater than about 70%;greater than about 80%; greater than about 90%; and greater than about95%, 98%, or 99%. In embodiments, the increase in activity is in anamount selected from the group consisting of: greater than about 5-fold;greater than about 8-fold; greater than about 10-fold. In embodiments,the increase in activity is in an amount selected from the groupconsisting of: greater than about 3-fold and greater than about 6-fold.

A method for identifying polypeptides that increase the flux in an Fe—Scluster biosynthesis pathway in a host cell comprising: changing theexpression or activity of a polypeptide affecting Fe—S clusterbiosynthesis; measuring the activity of a heterologous Fe—S clusterrequiring protein; and comparing the activity of the heterologous Fe—Scluster requiring protein measured in the presence of the change inexpression or activity of a polypeptide to the activity of theheterologous Fe—S cluster requiring protein measured in the absence ofthe change in expression or activity of a polypeptide, wherein anincrease in the activity of the heterologous Fe—S cluster requiringprotein indicates an increase in the flux in said Fe—S clusterbiosynthesis pathway.

Provided herein are methods for identifying polypeptides that increasethe flux in an Fe—S cluster biosynthesis pathway in a host cellcomprising: changing the expression or activity of a polypeptideaffecting Fe—S cluster biosynthesis; measuring the activity of apolypeptide having dihydroxy-acid dehydratase activity; and comparingthe activity of the polypeptide having dihydroxy-acid dehydrataseactivity measured in the presence of the change to the activity of thepolypeptide having dihydroxy-acid dehydratase activity measured in theabsence of change, wherein an increase in the activity of thepolypeptide having dihydroxy-acid dehydratase activity indicates anincrease in the flux in said Fe—S cluster biosynthesis pathway.

In embodiments, said changing the expression or activity of apolypeptide affecting Fe—S cluster biosynthesis comprises deleting,mutating, substituting, expressing, up-regulating, down-regulating,altering the cellular location, altering the state of the protein,and/or adding a cofactor. In embodiments, the Fe—S cluster requiringprotein has dihydroxy-acid dehydratase activity and wherein said Fe—Scluster requiring protein having dihydroxy-acid dehydratase activity hasan amino acid sequence that matches the Profile HMM of Table 12 with anE value of <10⁻⁵ wherein the polypeptide further comprises all threeconserved cysteines, corresponding to positions 56, 129, and 201 in theamino acids sequences of the Streptococcus mutans DHAD enzymecorresponding to SEQ ID NO:168. In embodiments, the polypeptideaffecting Fe—S cluster biosynthesis is selected from the groupconsisting of the genes in Tables 7, 8 and 9.

Also provided are recombinant host cells comprising at least onepolynucleotide encoding a polypeptide identified by the methods providedherein. In embodiments, said host cell further comprises at least oneheterologous polynucleotide encoding a polypeptide having dihydroxy-aciddehydratase activity. In embodiments, said heterologous polynucleotideencoding a polypeptide having dihydroxy-acid dehydratase activity isexpressed in multiple copies. In embodiments, said heterologouspolynucleotide comprises a high copy number plasmid or a plasmid with acopy number that can be regulated. In embodiments, said heterologouspolynucleotide is integrated at least once in the recombinant host cellDNA.

In embodiments, said host cell is a yeast host cell. In embodiments,said yeast host cell is selected from the group consisting ofSaccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces,Yarrowia, Issatchenkia, and Pichia. In embodiments, said heterologouspolypeptide having dihydroxy-acid dehydratase activity is expressed inthe cytosol of the host cell. In embodiments, said heterologouspolypeptide having dihydroxy-acid dehydratase activity has an amino acidsequence that matches the Profile HMM of Table 12 with an E value of<10⁻⁵ wherein the polypeptide further comprises all three conservedcysteines, corresponding to positions 56, 129, and 201 in the aminoacids sequences of the Streptococcus mutans DHAD enzyme corresponding toSEQ ID NO:168. In embodiments, said recombinant host cell produces aproduct selected from the group consisting of branched chain aminoacids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol,isobutanol, and combinations thereof. In embodiments, recombinant hostcell produces isobutanol. In embodiments, said recombinant host cellcomprises an isobutanol biosynthetic pathway. In embodiments saidisobutanol biosynthetic pathway comprises at least one polypeptideencoded by a polynucleotide heterologous to the host cell. Inembodiments, said isobutanol biosynthetic pathway comprises at least twopolypeptides encoded by polynucleotides heterologous to the host cell.

In embodiments, monomers of the polypeptides of the invention havingdihydroxy-acid dehydratase activity have an Fe—S cluster loadingselected from the group consisting of: (a) at least about 10%; (b) atleast about 15%; (c) at least about 20%; (d) at least about 25%; (e) atleast about 30%; (f) at least about 35%; (g) at least about 40%; (h) atleast about 45%; (i) at least about 50%; (j) at least about 60%; (k) atleast about 70%; (l) at least about 80%; (m) at least about 90%; and (n)at least about 95%.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A depicts a vector map of a vector for overexpression of the IlvDgene from S. mutans.

FIG. 1B depicts a vector map of an integration vector for overexpressionof the IlvD gene from S. mutans in the chromosome.

FIG. 2 depicts a vector map of a centromere vector used to clone AFT1 orAFT1 mutants and useful for other genes of interest.

FIG. 3 depicts a UV-Vis absorbance spectrum of purified S. mutans DHAD.

FIG. 4 depicts an EPR spectrum of purified S. mutans DHAD.

FIG. 5 depicts a biosynthetic pathway for biosynthesis of isobutanol.

FIG. 6A depicts a schematic of Azotobacter vinelandii nif genes.

FIG. 6B depicts a schematic of additional Azotobacter vinelandii nifgenes.

FIG. 6C depicts a schematic of the equation in which NFU acts as apersulfide reductase.

FIG. 7 depicts a schematic of Helicobacter pylori nif genes.

FIG. 8 depicts a schematic of E. coli isc genes.

FIG. 9 depicts a schematic of E. coli suf genes.

FIG. 10 depicts a schematic of the cytosolic [2Fe-2S] biosynthesis andassembly system.

FIG. 11 depicts a vector map of a vector for overexpression of the IlvDgene from L. lactis.

Table 12 is a table of the Profile HMM for dihydroxy-acid dehydratasesbased on enzymes with assayed function prepared as described in U.S.patent application Ser. No. 12/569,636, filed Sep. 29, 2009. Table 12 issubmitted herewith electronically and is incorporated herein byreference.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a method to increase the fraction of the Fe—Scluster requiring proteins that are loaded with Fe—S clusters. Alsodescribed are recombinant host cells that express functional Fe—Scluster requiring proteins, such as DHAD enzymes, and at least oneheterologous Fe uptake, utilization, or Fe—S cluster biosynthesisprotein, recombinant host cells that express functional DHAD enzymes andcomprise at least one deletion, mutation, and/or substitution in anative protein involved in Fe utilization or Fe—S cluster biosynthesis,or recombinant host cells comprising combinations thereof. In addition,the present invention describes a method to identify polypeptides thatincrease the flux in an Fe—S cluster biosynthesis pathway in a hostcell. Also described is a method to identify polypeptides that alter theactivity of an Fe—S cluster requiring protein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes.

In order to further define this invention, the following terms anddefinitions are herein provided.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers. For example, a composition, a mixture, a process,a method, an article, or an apparatus that comprises a list of elementsis not necessarily limited to only those elements but may include otherelements not expressly listed or inherent to such composition, mixture,process, method, article, or apparatus. Further, unless expressly statedto the contrary, “or” refers to an inclusive or and not to an exclusiveor. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present).

As used herein, the term “consists of” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers may be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition. See M.P.E.P. § 2111.03.

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the application.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates orsolutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathwayto produce isobutanol from pyruvate.

The term “a facultative anaerobe” refers to a microorganism that cangrow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof.

The term “Fe—S cluster biosynthesis” refers to biosynthesis of Fe—Sclusters, including, e.g., the assembly and loading of Fe—S clusters.The term “Fe—S cluster biosynthesis genes”, “Fe—S cluster biosynthesisproteins” or “Fe—S cluster biosynthesis pathway” refers to thosepolynucleotides/genes and the encoded polypeptides that are involved inthe biosynthesis of Fe—S clusters, including, e.g., the assembly andloading of Fe—S clusters.

The term “Fe uptake and utilization” refers to processes which caneffect Fe—S cluster biosynthesis such as Fe sensing, uptake,utilization, and homeostasis. “Fe uptake and utilization genes” refersto those polynucleotides/genes and the encoded polypeptides that areinvolved in Fe uptake, utilization, and homeostasis. Some of thesepolynucleotides/genes are contained in the “Fe Regulon” that has beendescribed in the literature and is further described hereafter. As usedherein, Fe uptake and utilization genes and Fe—S cluster biosynthesisgenes can encode a polypeptide affecting Fe—S cluster biosynthesis.

The term “specific activity” as used herein is defined as the units ofactivity in a given amount of protein. Thus, the specific activity isnot directly measured but is calculated by dividing 1) the activity inunits/ml of the enzyme sample by 2) the concentration of protein in thatsample, so the specific activity is expressed as units/mg. The specificactivity of a sample of pure, fully active enzyme is a characteristic ofthat enzyme. The specific activity of a sample of a mixture of proteinsis a measure of the relative fraction of protein in that sample that iscomposed of the active enzyme of interest. The specific activity of apolypeptide of the invention may be selected from greater than about0.25 U/mg; greater than about 0.3 U/mg; greater than about 0.4 U/mg;greater than about 0.5 U/mg; greater than about 0.6 U/mg; greater thanabout 0.7 U/mg; greater than about 0.8 U/mg; greater than about 0.9U/mg; greater than about 1.0 U/mg; greater than about 1.5 U/mg; greaterthan about 2.0 U/mg; greater than about 2.5 U/mg; greater than about 3.0U/mg; greater than about 3.5 U/mg; greater than about 4.0 U/mg; greaterthan about 5.5 U/mg; greater than about 5.0 U/mg; greater than about 6.0U/mg; greater than about 6.5 U/mg; greater than about 7.0 U/mg; greaterthan about 7.5 U/mg; greater than about 8.0 U/mg; greater than about 8.5U/mg; greater than about 9.0 U/mg; greater than about 9.5 U/mg; greaterthan about 10.0 U/mg; greater than about 20.0 U/mg; or greater thanabout 50.0 U/mg. In one embodiment, the specific activity of apolypeptide of the invention is greater than about 0.25 U/mg. In anotherembodiment, the specific activity is greater than about 1.0 U/mg. In yetanother embodiment, the specific activity is greater than about 2.0 U/mgor greater than about 3.0 U/mg.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to a nucleic acidmolecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).A polynucleotide can contain the nucleotide sequence of the full-lengthcDNA sequence, or a fragment thereof, including the untranslated 5′ and3′ sequences and the coding sequences. The polynucleotide can becomposed of any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. “Polynucleotide” embraceschemically, enzymatically, or metabolically modified forms.

A polynucleotide sequence may be referred to as “isolated,” in which ithas been removed from its native environment. For example, aheterologous polynucleotide encoding a polypeptide or polypeptidefragment having dihydroxy-acid dehydratase activity contained in avector is considered isolated for the purposes of the present invention.Further examples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. An isolatedpolynucleotide fragment in the form of a polymer of DNA may be comprisedof one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a polynucleotide that is capable of beingexpressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions.

The term “endogenous,” when used in reference to a polynucleotide, agene, or a polypeptide refers to a native polynucleotide or gene in itsnatural location in the genome of an organism, or for a nativepolypeptide, is transcribed and translated from this location in thegenome.

The term “heterologous” when used in reference to a polynucleotide, agene, or a polypeptide refers to a polynucleotide, gene, or polypeptidenot normally found in the host organism. “Heterologous” also includes anative coding region, or portion thereof, that is reintroduced into thesource organism in a form that is different from the correspondingnative gene, e.g., not in its natural location in the organism's genome.The heterologous polynucleotide or gene may be introduced into the hostorganism by, e.g., gene transfer. A heterologous gene may include anative coding region with non-native regulatory regions that isreintroduced into the native host. A “transgene” is a gene that has beenintroduced into the genome by a transformation procedure.

The term “recombinant genetic expression element” refers to a nucleicacid fragment that expresses one or more specific proteins, includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ termination sequences) coding sequences for the proteins. A chimericgene is a recombinant genetic expression element. The coding regions ofan operon may form a recombinant genetic expression element, along withan operably linked promoter and termination region.

“Regulatory sequences” refers to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, enhancers,operators, repressors, transcription termination signals, translationleader sequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure.

The term “promoter” refers to a nucleic acid sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic nucleic acid segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental or physiological conditions. Promoters which cause a geneto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. “Inducible promoters,” on the otherhand, cause a gene to be expressed when the promoter is induced orturned on by a promoter-specific signal or molecule. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andaccumulation of sense (mRNA) or antisense RNA derived from the nucleicacid fragment of the invention. Expression may also refer to translationof mRNA into a polypeptide. The process includes any manifestation ofthe functional presence of the expressed polynucleotide, gene, orpolypeptide within the cell including, without limitation, geneknockdown as well as both transient expression and stable expression.

The term “over-expression”, as used herein, refers to expression that ishigher than endogenous expression of the same or related polynucleotideor gene. A heterologous polynucleotide or gene is also over-expressed ifits expression is higher than that of a comparable endogenous gene, orif its expression is higher than that of the same polynucleotide or geneintroduced by a means that does not overexpress the polynucleotide orgene. For example, a polynucleotide can be expressed in a host cell froma low copy number plasmid, which is present in only limited or fewcopies, and the same polynucleotide can be over-expressed in a host cellfrom a high copy number plasmid or a plasmid with a copy number that canbe regulated, which is present in multiple copies. Any means can be usedto over-express a polynucleotide, so long as it increases the copies ofthe polynucleotide in the host cell. In addition to using a high copynumber plasmid, or a plasmid with a copy number that can be regulated, apolynucleotide can be over-expressed by multiple chromosomalintegrations.

Expression or over-expression of a polypeptide of the invention in arecombinant host cell can be quantified according to any number ofmethods known to the skilled artisan and can be represented, e.g., by apercent of total cell protein. The percent of total protein can be anamount selected from greater than about 0.001% of total cell protein;greater than about 0.01% of total cell protein; greater than about 0.1%of total cell protein; greater than about 0.5% of total cell protein;greater than about 1.0% of total cell protein; greater than about 2.0%of total cell protein; greater than about 3% of total cell protein;greater than about 4.0% of total cell protein; greater than about 5% oftotal cell protein; greater than about 6.0% of total cell protein;greater than about 7.0% of total cell protein; greater than about 8.0%of total cell protein; greater than about 9.0% of total cell protein;greater than about 10% of total cell protein; or greater than about 20%of total cell protein. In one embodiment, the amount of polypeptideexpressed is greater that about 0.5% of total cell protein. In anotherembodiment, the amount of polypeptide expressed is greater than about1.0% of total cell protein or greater than about 2.0% of total cellprotein.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance with or without selections. Host organisms containingthe transformed nucleic acid fragments are referred to as “transgenic”or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withouteffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S)TAT Tyr (Y) TGT Cys (C) TTC″ TCC″ TAC″ TGC TTA Leu (L) TCA″ TAA StopTGA Stop TTG″ TCG″ TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P)CAT His (H) CGT Arg (R) CTC″ CCC″ CAC″ CGC″ CTA″ CCA″ CAA Gln (Q) CGA″CTG″ CCG″ CAG″ CGG″ A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S)ATC″ ACC″ AAC″ AGC″ ATA″ ACA″ AAA Lys (K) AGA Arg (R) ATG Met ACG″ AAG″AGG″ (M) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC″ GCC″GAC″ GGC″ GTA″ GCA″ GAA Glu (E) GGA″ GTG″ GCG″ GAG″ GGG″

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available at theKazusa DNA Research Institute, Japan, and these tables can be adapted ina number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292(2000). Codon usage tables for yeast, calculated from GenBank Release128.0 [15 Feb. 2002], are reproduced below as Table 2. This table usesmRNA nomenclature, and so instead of thymine (T) which is found in DNA,the tables use uracil (U) which is found in RNA. Table 2 has beenadapted so that frequencies are calculated for each amino acid, ratherthan for all 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Frequencyper Amino Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 12051018.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 IleAUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 14424322.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 SerAGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 8320712.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 TyrUAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 310954.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 195623.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 StopUAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTI Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function(Entelechon GmbH, Regensburg, Germany) and the “backtranseq” function(NRC Saskatoon Bioinformatics, Saskatoon, Saskatchewan, Canada).Constructing a rudimentary algorithm to assign codons based on a givenfrequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods knownto those skilled in the art including software packages such as“synthetic gene designer” (University of Maryland, Baltimore, Md.).

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the term “variant” refers to a polypeptide differingfrom a specifically recited polypeptide of the invention, such as DHAD,by amino acid insertions, deletions, mutations, and substitutions,created using, e.g., recombinant DNA techniques, such as mutagenesis.Guidance in determining which amino acid residues may be replaced,added, or deleted without abolishing activities of interest, may befound by comparing the sequence of the particular polypeptide with thatof homologous polypeptides, e.g., yeast or bacterial, and minimizing thenumber of amino acid sequence changes made in regions of high homology(conserved regions) or by replacing amino acids with consensussequences.

Alternatively, recombinant polynucleotide variants encoding these sameor similar polypeptides may be synthesized or selected by making use ofthe “redundancy” in the genetic code. Various codon substitutions, suchas silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector forexpression. Mutations in the polynucleotide sequence may be reflected inthe polypeptide or domains of other peptides added to the polypeptide tomodify the properties of any part of the polypeptide. For example,mutations can be used to reduce or eliminate expression of a targetprotein and include, but are not limited to, deletion of the entire geneor a portion of the gene, inserting a DNA fragment into the gene (ineither the promoter or coding region) so that the protein is notexpressed or expressed at lower levels, introducing a mutation into thecoding region which adds a stop codon or frame shift such that afunctional protein is not expressed, and introducing one or moremutations into the coding region to alter amino acids so that anon-functional or a less enzymatically active protein is expressed.

Amino acid “substitutions” may be the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, i.e., conservative amino acid replacements, or they may bethe result of replacing one amino acid with an amino acid havingdifferent structural and/or chemical properties, i.e., non-conservativeamino acid replacements. “Conservative” amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. Alternatively, “non-conservative” amino acidsubstitutions may be made by selecting the differences in polarity,charge, solubility, hydrophobicity, hydrophilicity, or the amphipathicnature of any of these amino acids. “Insertions” or “deletions” may bewithin the range of variation as structurally or functionally toleratedby the recombinant proteins. The variation allowed may be experimentallydetermined by systematically making insertions, deletions, orsubstitutions of amino acids in a polypeptide molecule using recombinantDNA techniques and assaying the resulting recombinant variants foractivity.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten ormore contiguous amino acids or thirty or more nucleotides is necessaryin order to putatively identify a polypeptide or nucleic acid sequenceas homologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenine is complementary to thymine andcytosine is complementary to guanine, and with respect to RNA, adenineis complementary to uracil and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences is performed using the “Clustal method of alignment” whichencompasses several varieties of the algorithm including the “Clustal Vmethod of alignment” corresponding to the alignment method labeledClustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989);Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) andfound in the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc.). For multiple alignments, the default valuescorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After alignment of the sequences using the Clustal V program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program. Additionally the “Clustal W methodof alignment” is available and corresponds to the alignment methodlabeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992))and found in the MegAlign™ v6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc.). Default parameters for multiplealignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay DivergenSeqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=GonnetSeries, DNA Weight Matrix=IUB). After alignment of the sequences usingthe Clustal W program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity, or in describing the corresponding polynucleotides. Usefulexamples of percent identities include, but are not limited to: 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentagefrom 55% to 100% may be useful in describing the present invention, suchas 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%. Suitable polynucleotide fragments not only have theabove homologies but typically comprise a polynucleotide having at least50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, atleast 200 nucleotides, or at least 250 nucleotides. Further, suitablepolynucleotide fragments having the above homologies encode apolypeptide having at least 50 amino acids, at least 100 amino acids, atleast 150 amino acids, at least 200 amino acids, or at least 250 aminoacids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)SEQUENCHER (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

The Functions of Fe—S Cluster-Requiring Proteins

The functions of proteins that contain Fe—S clusters are diverse. One ofthe more complete efforts to classify these functions is given in thefollowing table which is adapted from Johnson, D. C., et al., Structure,function, and formation of biological iron-sulfur clusters. Annu. Rev.Biochem., 2005. 74: p. 247-281.

TABLE 3 Functions of Biological [Fe—S] clusters^(a). Function ExamplesCluster type Electron transfer Ferredoxins; redox [2Fe—2S]; [3Fe—4S];[4Fe—4S] enzymes Coupled electron/proton Rieske protein [2Fe—2S]transfer Nitrogenase [8Fe—7S] Substrate binding and (de)Hydratases[4Fe—4S], [2Fe—2S] activation Radical SAM enzymes [4Fe—4S] Acetyl-CoAsynthase Ni—Ni—[4Fe—4S], [Ni—4Fe—5S] Sulfite reductase [4Fe—4S]-sirohemeFe or cluster storage Ferredoxins [4Fe—4S] Polyferredoxins [4Fe—4S]Structural Endonuclease III [4Fe—4S] MutY [4Fe—4S] Regulation of geneSoxR [2Fe—2S] expression FNR [4Fe—4S]/[2Fe—2S] IRP [4Fe—4S] IscR[2Fe—2S] Regulation of enzyme Glutamine PRPP [4Fe—4S] activityamidotransferase Ferrochelatase [2Fe—2S] Disulfide reductionFerredoxin:thioredoxin [4Fe—4S] reductase Heterodisulfide [4Fe—4S]reductase Sulfur donor Biotin synthase [2Fe—2S] ^(a)Abbreviations usedare SAM, S-adenosylmethionine; acetyl-CoA, acetyl coenzymeA; FNR,fumarate and nitrate reduction; IRP, iron-regulatory protein; IscR,iron-sulfur cluster assembly regulatory protein; PRPP,phosphoribosylpyrophosphate.

It is believed that an increase in the supply and the efficiency ofloading Fe—S clusters into one or more of the members of the aboveclasses will have commercial and/or medical benefits. Of the manypossibilities that will be appreciated by the skilled artisan, threeexamples are given. 1) When an Fe—S cluster containing enzyme is used ina pathway to a fermentation product and needs to be expressed at highlevels to maintain a high flux in the pathway to the product (e.g.,dihydroxy-acid dehydratase in the pathway to isobutanol). 2) When anFe—S cluster containing enzyme is used in a pathway to a fermentationproduct and the Fe—S cluster undergoes turnover during the catalysis(e.g., biotin synthase in the commercial fermentation of glucose tobiotin). 3) In a diseased state such that the normal concentration of anFe—S cluster containing protein important for good health is low (e.g.,in cases of Friedreich's ataxia).

DHAD and DHAD Assays

DHAD is an Fe—S cluster requiring protein of the dehydratase (moreproperly hydro-lyase) class. A gene encoding a DHAD enzyme can be usedto provide expression of DHAD activity in a recombinant host cell. DHADcatalyzes the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate and of 2,3-dihydroxymethylvalerate toα-ketomethylvalerate and is classified as E.C. 4.2.1.9. Coding sequencesfor DHADs that are suitable for use in a recombinant host cell can bederived from bacterial, fungal, or plant sources. DHADs that may be usedmay have a [4Fe-4S] cluster or a [2Fe-2S]. Tables 4a, 4b, 5, and 6 listSEQ ID NOs for coding regions and proteins of representative DHADs thatmay be used in the present invention. Proteins with at least about 95%identity to certain listed sequences have been omitted forsimplification, but it is understood that proteins, including thoseomitted for simplification, with at least about 95% sequence identity toany of the proteins listed in Tables 4a, 4b, 5, and 6 and having DHADactivity may be used as disclosed herein. Additional DHAD proteins andtheir encoding sequences may be identified by BLAST searching of publicdatabases, as well known to one skilled in the art. Typically BLAST(described above) searching of publicly available databases with knownDHAD sequences, such as those provided herein, is used to identify DHADsand their encoding sequences that may be expressed in the present cells.For example, DHAD proteins having amino acid sequence identities of atleast about 80-85%, at least about 85-90%, at least about 90-95%, or atleast about 98% sequence identity to any of the DHAD proteins of Table 3may be expressed in the present cells. Identities are based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.

TABLE 4a SEQ ID NOs of Representative Bacterial [2Fe—2S] DHAD Proteinsand Encoding Sequences. SEQ ID NO: SEQ Nucleic ID NO: Organism ofderivation acid Peptide Mycobacterium sp. MCS 1 2 Mycobacterium gilvumPYR-GCK 3 4 Mycobacterium smegmatis str. MC2 155 5 6 Mycobacteriumvanbaalenii PYR-1 7 8 Nocardia farcinica IFM 10152 9 10 Rhodococcus sp.RHA1 11 12 Mycobacterium ulcerans Agy99 13 14 Mycobacterium avium subsp.paratuberculosis K-10 15 16 Mycobacterium tuberculosis H37Ra 17 18Mycobacterium leprae TN * 19 20 Kineococcus radiotolerans SRS30216 21 22Janibacter sp. HTCC2649 23 24 Nocardioides sp. JS614 25 26 Renibacteriumsalmoninarum ATCC 33209 27 28 Arthrobacter aurescens TC1 29 30 Leifsoniaxyli subsp. xyli str. CTCB07 31 32 marine actinobacterium PHSC20C1 33 34Clavibacter michiganensis subsp. michiganensis 35 36 NCPPB 382Saccharopolyspora erythraea NRRL 2338 37 38 Acidothermus cellulolyticus11B 39 40 Corynebacterium efficiens YS-314 41 42 Brevibacterium linensBL2 43 44 Tropheryma whipplei TW08/27 45 46 Methylobacterium extorquensPA1 47 48 Methylobacterium nodulans ORS 2060 49 50 Rhodopseudomonaspalustris BisB5 51 52 Rhodopseudomonas palustris BisB18 53 54Bradyrhizobium sp. ORS278 55 56 Bradyrhizobium japonicum USDA 110 57 58Fulvimarina pelagi HTCC2506 59 60 Aurantimonas sp. SI85-9A1 61 62Hoeflea phototrophica DFL-43 63 64 Mesorhizobium loti MAFF303099 65 66Mesorhizobium sp. BNC1 67 68 Parvibaculum lavamentivorans DS-1 69 70Loktanella vestfoldensis SKA53 71 72 Roseobacter sp. CCS2 73 74Dinoroseobacter shibae DFL 12 75 76 Roseovarius nubinhibens ISM 77 78Sagittula stellata E-37 79 80 Roseobacter sp. AzwK-3b 81 82 Roseovariussp. TM1035 83 84 Oceanicola batsensis HTCC2597 85 86 Oceanicolagranulosus HTCC2516 87 88 Rhodobacterales bacterium HTCC2150 89 90Paracoccus denitrificans PD1222 91 92 Oceanibulbus indolifex HEL-45 9394 Sulfitobacter sp. EE-36 95 96 Roseobacter denitrificans OCh 114 97 98Jannaschia sp. CCS1 99 100 Caulobacter sp. K31 101 102 CandidatusPelagibacter ubique HTCC1062 103 104 Erythrobacter litoralis HTCC2594105 106 Erythrobacter sp. NAP1 107 108 Comamonas testosterone KF-1 109110 Sphingomonas wittichii RW1 111 112 Burkholderia xenovorans LB400 113114 Burkholderia phytofirmans PsJN 115 116 Bordetella petrii DSM 12804117 118 Bordetella bronchiseptica RB50 119 120 Bradyrhizobium sp. ORS278121 122 Bradyrhizobium sp. BTAi1 123 124 Bradhyrhizobium japonicum 125126 Sphingomonas wittichii RW1 127 128 Rhodobacterales bacteriumHTCC2654 129 130 Solibacter usitatus Ellin6076 131 132 Roseiflexus sp.RS-1 133 134 Rubrobacter xylanophilus DSM 9941 135 136 Salinisporatropica CNB-440 137 138 Acidobacteria bacterium Ellin345 139 140 Thermusthermophilus HB27 141 142 Maricaulis maris MCS10 143 144 Parvularculabermudensis HTCC2503 145 146 Oceanicaulis alexandrii HTCC2633 147 148Plesiocystis pacifica SIR-1 149 150 Bacillus sp. NRRL B-14911 151 152Oceanobacillus iheyensis HTE831 153 154 Staphylococcus saprophyticussubsp. saprophyticus 155 156 ATCC 15305 Bacillus selenitireducens MLS10157 158 Streptococcus pneumoniae SP6-BS73 159 160 Streptococcussanguinis SK36 161 162 Streptococcus thermophilus LMG 18311 163 164Streptococcus suis 89/1591 165 166 Streptococcus mutans UA159 167 168Leptospira borgpetersenii serovar Hardjo-bovis L550 169 170 CandidatusVesicomyosocius okutanii HA 171 172 Candidatus Ruthia magnifica str. Cm(Calyptogena 173 174 magnifica) Methylococcus capsulatus str. Bath 175176 uncultured marine bacterium EB80_02D08 177 178 uncultured marinegamma proteobacterium 179 180 EBAC31A08 uncultured marine gammaproteobacterium 181 182 EBAC20E09 uncultured gamma proteobacteriumeBACHOT4E07 183 184 Alcanivorax borkumensis SK2 185 186 Chromohalobactersalexigens DSM 3043 187 188 Marinobacter algicola DG893 189 190Marinobacter aquaeolei VT8 191 192 Marinobacter sp. ELB17 193 194Pseudoalteromonas haloplanktis TAC125 195 196 Acinetobacter sp. ADP1 197198 Opitutaceae bacterium TAV2 199 200 Flavobacterium sp. MED217 201 202Cellulophaga sp. MED134 203 204 Kordia algicida OT-1 205 206Flavobacteriales bacterium ALC-1 207 208 Psychroflexus torquis ATCC700755 209 210 Flavobacteriales bacterium HTCC2170 211 212 unidentifiedeubacterium SCB49 213 214 Gramella forsetii KT0803 215 216 Robiginitaleabiformata HTCC2501 217 218 Tenacibaculum sp. MED152 219 220 Polaribacterirgensii 23-P 221 222 Pedobacter sp. BAL39 223 224 Flavobacteriabacterium BAL38 225 226 Flavobacterium psychrophilum JIP02/86 227 228Flavobacterium johnsoniae UW101 229 230 Lactococcus lactis subsp.cremoris SK11 231 232 Psychromonas ingrahamii 37 233 234 Microscillamarina ATCC 23134 235 236 Cytophaga hutchinsonii ATCC 33406 237 238Rhodopirellula baltica SH 1 239 240 Blastopirellula marina DSM 3645 241242 Planctomyces maris DSM 8797 243 244 Algoriphagus sp. PR1 245 246Candidatus Sulcia muelleri str. Hc (Homalodisca 247 248 coagulata)Candidatus Carsonella ruddii PV 249 250 Synechococcus sp. RS9916 251 252Synechococcus sp. WH 7803 253 254 Synechococcus sp. CC9311 255 256Synechococcus sp. CC9605 257 258 Synechococcus sp. WH 8102 259 260Synechococcus sp. BL107 261 262 Synechococcus sp. RCC307 263 264Synechococcus sp. RS9917 265 266 Synechococcus sp. WH 5701 267 268Prochlorococcus marinus str. MIT 9313 269 270 Prochlorococcus marinusstr. NATL2A 271 272 Prochlorococcus marinus str. MIT 9215 273 274Prochlorococcus marinus str. AS9601 275 276 Prochlorococcus marinus str.MIT 9515 277 278 Prochlorococcus marinus subsp. pastoris str. 279 280CCMP1986 Prochlorococcus marinus str. MIT 9211 281 282 Prochlorococcusmarinus subsp. marinus str. 283 284 CCMP1375 Nodularia spumigena CCY9414285 286 Nostoc punctiforme PCC 73102 287 288 Nostoc sp. PCC 7120 289 290Trichodesmium erythraeum IMS101 291 292 Acaryochloris marina MBIC11017293 294 Lyngbya sp. PCC 8106 295 296 Synechocystis sp. PCC 6803 297 298Cyanothece sp. CCY0110 299 300 Thermosynechococcus elongatus BP-1 301302 Synechococcus sp. JA-2-3B′a(2-13) 303 304 Gloeobacter violaceus PCC7421 305 306 Nitrosomonas eutropha C91 307 308 Nitrosomonas europaeaATCC 19718 309 310 Nitrosospira multiformis ATCC 25196 311 312Chloroflexus aggregans DSM 9485 313 314 Leptospirillum sp. Group II UBA315 316 Leptospirillum sp. Group II UBA 317 318 Halorhodospira halophilaSL1 319 320 Nitrococcus mobilis Nb-231 321 322 Alkalilimnicola ehrlicheiMLHE-1 323 324 Deinococcus geothermalis DSM 11300 325 326Polynucleobacter sp. QLW-P1DMWA-1 327 328 Polynucleobacter necessariusSTIR1 329 330 Azoarcus sp. EbN1 331 332 Burkholderia phymatum STM815 333334 Burkholderia xenovorans LB400 335 336 Burkholderia multivorans ATCC17616 337 338 Burkholderia cenocepacia PC184 339 340 Burkholderia malleiGB8 horse 4 341 342 Ralstonia eutropha JMP134 343 344 Ralstoniametallidurans CH34 345 346 Ralstonia solanacearum UW551 347 348Ralstonia pickettii 12J 349 350 Limnobacter sp. MED105 351 352Herminiimonas arsenicoxydans 353 354 Bordetella parapertussis 355 356Bordetella petrii DSM 12804 357 358 Polaromonas sp. JS666 359 360Polaromonas naphthalenivorans CJ2 361 362 Rhodoferax ferrireducens T118363 364 Verminephrobacter eiseniae EF01-2 365 366 Acidovorax sp. JS42367 368 Delftia acidovorans SPH-1 369 370 Methylibium petroleiphilum PM1371 372 gamma proteobacterium KT 71 373 374 Tremblaya princes 375 376Blastopirellula marina DSM 3645 377 378 Planctomyces maris DSM 8797 379380 Microcystis aeruginosa PCC 7806 381 382 Salinibacter ruber DSM 13855383 384 Methylobacterium chloromethanicum 385 386

TABLE 4B Additional representative bacterial [2Fe—2S] DHAD proteins andencoding sequences. Nucleic acid Amino acid Organism of derivation SEQID NO: SEQ ID NO: Burkholderia ambifaria AMMD 387 388 Bradyrhizobium sp.BTAi1 389 390 Delftia acidovorans SPH-1 391 392 Microcystis aeruginosaNIES-843 393 394 uncultured marine microorganism 395 396 HF4000_APKG8C21Burkholderia ubonensis Bu 397 398 Gemmata obscuriglobus UQM 2246 399 400Mycobacterium abscessus 401 402 Synechococcus sp. PCC 7002 403 404Burkholderia graminis C4D1M 405 406 Methylobacterium radiotolerans JCM2831 407 408 Leptothrix cholodnii SP-6 409 410 Verrucomicrobium spinosumDSM 4136 411 412 Cyanothece sp. ATCC 51142 413 414 Opitutus terraePB90-1 415 416 Leptospira biflexa serovar Patoc strain 417 418 ‘Patoc 1(Paris)’ Methylacidiphilum infernorum V4 419 420 Cupriavidus taiwanensis421 422 Chthoniobacter flavus Ellin428 423 424 Cyanothece sp. PCC 7822425 426 Phenylobacterium zucineum HLK1 427 428 Leptospirillum sp. GroupII ‘5-way CG’ 429 430 Arthrospira maxima CS-328 431 432 Oligotrophacarboxidovorans OM5 433 434 Rhodospirillum centenum SW 435 436Cyanothece sp. PCC 8801 437 438 Thermus aquaticus Y51MC23 439 440Cyanothece sp. PCC 7424 441 442 Acidithiobacillus ferrooxidans ATCC23270 443 444 Cyanothece sp. PCC 7425 445 446 Arthrobacterchlorophenolicus A6 447 448 Burkholderia multivorans CGD2M 449 450Thermomicrobium roseum DSM 5159 451 452 bacterium Ellin514 453 454Desulfobacterium autotrophicum HRM2 455 456 Thioalkalivibrio sp. K90mix457 458 Flavobacteria bacterium MS024-3C 459 460 Flavobacteria bacteriumMS024-2A 461 462 ‘Nostoc azollae’ 0708 463 464 Acidobacterium capsulatumATCC 51196 465 466 Gemmatimonas aurantiaca T-27 467 468 Gemmatimonasaurantiaca T-27 469 470 Rhodococcus erythropolis PR4 471 472 Deinococcusdeserti VCD115 473 474 Rhodococcus opacus B4 475 476 Chryseobacteriumgleum ATCC 35910 477 478 Thermobaculum terrenum ATCC BAA-798 479 480Kribbella flavida DSM 17836 481 482 Gordonia bronchialis DSM 43247 483484 Geodermatophilus obscurus DSM 43160 485 486 Xylanimonascellulosilytica DSM 15894 487 488 Sphingobacterium spiritivorum ATCC33300 489 490 Meiothermus silvanus DSM 9946 491 492 Meiothermus ruberDSM 1279 493 494 Nakamurella multipartita DSM 44233 495 496 Cellulomonasflavigena DSM 20109 497 498 Rhodothermus marinus DSM 4252 499 500Planctomyces limnophilus DSM 3776 501 502 Beutenbergia cavernae DSM12333 503 504 Spirosoma linguale DSM 74 505 506 Sphaerobacterthermophilus DSM 20745 507 508 Lactococcus lactis 509 510 Thermusthermophilus HB8 511 512 Anabaena variabilis ATCC 29413 513 514Roseovarius sp. 217 515 516 uncultured Prochlorococcus marinus 517 518clone HF10-88D1 Burkholderia xenovorans LB400 519 520 Saccharomonosporaviridis DSM 43017 521 522 Pedobacter heparinus DSM 2366 523 524Microcoleus chthonoplastes PCC 7420 525 526 Acidimicrobium ferrooxidansDSM 10331 527 528 Rhodobacterales bacterium HTCC2083 529 530 CandidatusPelagibacter sp. HTCC7211 531 532 Chitinophaga pinensis DSM 2588 533 534Alcanivorax sp. DG881 535 536 Micrococcus luteus NCTC 2665 537 538Verrucomicrobiae bacterium DG1235 539 540 Synechococcus sp. PCC 7335 541542 Brevundimonas sp. BAL3 543 544 Dyadobacter fermentans DSM 18053 545546 gamma proteobacterium NOR5-3 547 548 gamma proteobacterium NOR51-B549 550 Cyanobium sp. PCC 7001 551 552 Jonesia denitrificans DSM 20603553 554 Brachybacterium faecium DSM 4810 555 556 Paenibacillus sp. JDR-2557 558 Octadecabacter antarcticus 307 559 560 Variovorax paradoxus S110561 562

TABLE 5 SEQ ID NOs of Representative Fungal and Plant [2Fe—2S] DHADProteins and Encoding Sequences. SEQ ID NO: SEQ ID NO: DescriptionNucleic acid Peptide Schizosaccharomyces pombe ILV3 563 564Saccharomyces cerevisiae ILV3 565 566 Kluyveromyces lactis ILV3 567 568Candida albicans SC5314 ILV3 569 570 Pichia stipitis CBS 6054 ILV3 571572 Yarrowia lipolytica ILV3 573 574 Candida galbrata CBS 138 ILV3 575576 Chlamydomonas reinhardtii 577 578 Ostreococcus lucimarinus CCE9901579 580 Vitis vinifera 581 582 (Unnamed protein product: CAO71581.1)Vitis vinifera 583 584 (Hypothetical protein: CAN67446.1) Arabidopsisthaliana 585 586 Oryza sativa (indica cultivar-group) 587 588Physcomitrella patens subsp. Patens 589 590 Chaetomium globosum CBS148.51 591 592 Neurospora crassa OR74A 593 594 Magnaporthe grisea 70-15595 596 Gibberella zeae PH-1 597 598 Aspergillus niger 599 600Neosartorya fischeri NRRL 181 601 602 (XP_001266525.1) Neosartoryafischeri NRRL 181 603 604 (XP_001262996.1) Aspergillus niger 605 606(hypothetical protein An03g04520) Aspergillus niger 607 608(Hypothetical protein An14g03280) Aspergillus terreus NIH2624 609 610Aspergillus clavatus NRRL 1 611 612 Aspergillus nidulans FGSC A4 613 614Aspergillus oryzae 615 616 Ajellomyces capsulatus NAm1 617 618Coccidioides immitis RS 619 620 Botryotinia fuckeliana B05.10 621 622Phaeosphaeria nodorum SN15 623 624 Pichia guilliermondii ATCC 6260 625626 Debaryomyces hansenii CBS767 627 628 Lodderomyces elongisporus NRRLYB-4239 629 630 Vanderwaltozyma polyspora DSM 70294 631 632 Ashbyagossypii ATCC 10895 633 634 Laccaria bicolor S238N-H82 635 636Coprinopsis cinerea okayama 7#130 637 638 Cryptococcus neoformans var.639 640 neoformans JEC21 Ustilago maydis 521 641 642 Malassezia globosaCBS 7966 643 644 Aspergillus clavatus NRRL 1 645 646 Neosartoryafischeri NRRL 181 647 648 (Putative) Aspergillus oryzae 649 650Aspergillus niger (hypothetical 651 652 protein An18g04160) Aspergillusterreus NIH2624 653 654 Coccidioides immitis RS (hypothetical 655 656protein CIMG_04591) Paracoccidioides brasiliensis 657 658 Phaeosphaerianodorum SN15 659 660 Gibberella zeae PH-1 661 662 Neurospora crassaOR74A 663 664 Coprinopsis cinerea okayama 7#130 665 666 Laccaria bicolorS238N-H82 667 668 Ustilago maydis 521 669 670

TABLE 6 SEQ ID NOs of Representative [4Fe—4S] DHAD Proteins and EncodingSequences. SEQ ID NO: SEQ ID NO: Organism Nucleic acid PeptideEscherichia coli str. K-12 substr. MG1655 671 672 Bacillus subtilissubsp. subtilis str. 168 673 674 Agrobacterium tumefaciens str. C58 675676 Burkholderia cenocepacia MC0-3 677 678 Psychrobacter cryohalolentisK5 679 680 Psychromonas sp. CNPT3 681 682 Deinococcus radiodurans R1 683684 Wolinella succinogenes DSM 1740 685 686 Zymomonas mobilis subsp.mobilis ZM4 687 688 Clostridium acetobutylicum ATCC 824 689 690Clostridium beijerinckii NCIMB 8052 691 692 Pseudomonas fluorescens Pf-5693 694 Methanococcus maripaludis C7 695 696 Methanococcus aeolicusNankai-3 697 698 Vibrio fischeri ATCC 700601 (ES114) 699 700 Shewanellaoneidensis MR-1 ATCC 700550 701 702

Additional [2Fe-2S] DHADs may be identified using the analysis describedin U.S. patent application Ser. No. 12/569,636, filed Sep. 29, 2009,which is herein incorporated by reference. The analysis is as follows: AProfile Hidden Markov Model (HMM) was prepared based on amino acidsequences of eight functionally verified DHADs. The application ofProfile HMM has been described. See, e.g., Krogh et al., J. Mol. Biol.235:1501-1531 (1994) and Durbin et al., “Markov chains and hidden Markovmodels,” in Biological Sequence Analysis: Probabilistic Models ofProteins and Nucleic Acids, Cambridge University Press (1998). A ProfileHMM is a statistical model built of multiple sequence alignments thatcan be used to determine whether or not a test sequence belongs to aparticular family of sequences. See id. A Profile HMM can be built byfirst generating an alignment of functionally verified sequences usingconventional sequence alignment tools. Next, the sequence alignment isused to build the Profile HMM using publicly available software programs(e.g., HMMER) that use a position-specific scoring system to captureinformation about the degree of conservation at various amino acidpositions in the multiple alignment of the input sequences. Morespecifically, the scores of amino acid residues in a “match” state(i.e., match state emission scores), or in an “insert” state (i.e.,insert state emission scores) are captured which are proportional to theexpression: Log_2 (p_x)/(null_x). See id. In this expression, the term“p_x” is the probability of an amino acid residue, at a particularposition in the alignment, according to the Profile HMM, and the term“null_x” is the probability according to the Null model. See id. TheNull model is a simple one state probabilistic model with apre-calculated set of emission probabilities for each of the amino acidsderived from the distribution of amino acids. See id. “State” transitionscores are also calculated as log odds parameters and are proportionalto Log_2 (t_x). See id. In this expression, the term “t_x” is theprobability of transiting to an emitter or non-emitter state. See id.Further details regarding the particular statistical analyses togenerate a Profile HMM are available in Krogh et al., J. Mol. Biol.235:1501-1531 (1994) and Durbin et al., “Markov chains and hidden Markovmodels,” in Biological Sequence Analysis: Probabilistic Models ofProteins and Nucleic Acids, Cambridge University Press (1998), and U.S.patent application Ser. No. 12/569,636.

A Profile Hidden Markov Model (HMM) was prepared based on amino acidsequences of eight functionally verified DHADs are from Nitrosomonaseuropaea (DNA SEQ ID NO:309; protein SEQ ID NO:310), Synechocystis sp.PCC6803 (DNA SEQ ID:297; protein SEQ ID NO:298), Streptococcus mutans(DNA SEQ ID NO:167; protein SEQ ID NO:168), Streptococcus thermophilus(DNA SEQ ID NO:163; SEQ ID No:164), Ralstonia metallidurans (DNA SEQ IDNO:345; protein SEQ ID NO:346), Ralstonia eutropha (DNA SEQ ID NO:343;protein SEQ ID NO:344), and Lactococcus lactis (DNA SEQ ID NO:231;protein SEQ ID NO:232). In addition the DHAD from Flavobacteriumjohnsoniae (DNA SEQ ID NO:229; protein SEQ ID NO:230) was found to havedihydroxy-acid dehydratase activity when expressed in E. coli and wasused in making the Profile. The Profile HMM is prepared using the HMMERsoftware package (The theory behind profile HMMs is described in R.Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequenceanalysis: probabilistic models of proteins and nucleic acids, CambridgeUniversity Press, 1998; Krogh et al., 1994; J. Mol. Biol.235:1501-1531), following the user guide which is available from HMMER(Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMERsoftware program is a Profile Hidden Markov Model (HMM) thatcharacterizes the input sequences. The Profile HMM prepared for theeight DHAD proteins is given in U.S. application Ser. No. 12/569,636,filed Sep. 29, 2009 and in Table 12.

The first line in Table 12 for each position reports the probability foreach amino acid to be in that “state” (match state emission scores). Thesecond line reports the insert state emission scores, and the third linereports the state transition scores. The highest probability ishighlighted for each position. These scores can be converted into “Evalues” (expectation values), which are the number of hits or matches tothe Profile HMM one would expect to obtain just by chance. A proteinhaving an E value of <10⁻⁵ match to the Profile HMM, indicates that theprotein shares significant sequence similarity with the seed proteinsused to construct the Profile HMM and that the protein belongs to thefamily represented by the profile HMM.

Any protein that matches the Profile HMM with an E value of <10⁻⁵ is aDHAD related protein, which includes [4Fe-4S] DHADs, [2Fe-2S] DHADs,arabonate dehydratases, and phosphogluconate dehydratases. Inembodiments, sequences matching the Profile HMM are then analyzed forthe presence of the three conserved cysteines, corresponding topositions 56, 129, and 201 in the Streptococcus mutans DHAD. Thepresence of all three conserved cysteines is characteristic of proteinshaving a [2Fe-2S] cluster. Proteins having the three conserved cysteinesinclude arabonate dehydratases and [2Fe-2S] DHADs. The [2Fe-2S] DHADsmay be distinguished from the arabonate dehydratases by analyzing forsignature conserved amino acids found to be present in the [2Fe-2S]DHADs or in the arabonate dehydratases at positions corresponding to thefollowing positions in the Streptococcus mutans DHAD amino acidsequence. These signature amino acids are in [2Fe-2S] DHADs or inarabonate dehydratases, respectively, at the following positions (withgreater than 90% occurrence): 88 asparagine vs. glutamic acid; 113 notconserved vs. glutamic acid; 142 arginine or asparagine vs. notconserved; 165 not conserved vs. glycine; 208 asparagine vs. notconserved; 454 leucine vs. not conserved; 477 phenylalanine or tyrosinevs. not conserved; and 487 glycine vs. not conserved.

Additionally, the sequences of DHAD coding regions provided herein maybe used to identify other homologs in nature. Such methods arewell-known in the art, and various methods that may be used to isolategenes encoding homologous proteins are described in U.S. applicationSer. No. 12/569,636, filed Sep. 29, 2009, which such methods areincorporated by reference herein.

The presence of DHAD activity in a cell engineered to express aheterologous DHAD can be confirmed using methods known in the art. Asone example, and as demonstrated in the Examples herein, crude extractsfrom cells engineered to express a bacterial DHAD may be used in a DHADassay as described by Flint and Emptage (J. Biol. Chem. (1988) 263(8):3558-64) using dinitrophenylhydrazine. In another example, DHAD activitymay be assayed by expressing a heterologous DHAD identifiable by themethods disclosed herein in a yeast strain that lacks endogenous DHADactivity. If DHAD activity is present, the yeast strain will grow in theabsence of branched-chain amino acids. DHAD activity may also beconfirmed by more indirect methods, such as by assaying for a downstreamproduct in a pathway requiring DHAD activity. Any product that hasα-ketoisovalerate or α-ketomethylvalerate as a pathway intermediate maybe measured in an assay for DHAD activity. A list of such productsincludes, but is not limited to, valine, isoleucine, leucine,pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, andisobutanol.

Over-Expression of DHAD Activity

Applicants have found that expression of a heterologous DHAD can provideDHAD activity when expressed in a host cell. Expression of a DHAD whichmay be identified as described herein can provide DHAD activity for abiosynthetic pathway that includes conversion of2,3-dihydroxyisovalerate to α-ketoisovalerate or2,3-dihydroxymethylvalerate to α-ketomethylvalerate. In addition, the S.mutans [2Fe-2S] DHAD was shown in related U.S. application Ser. No.12/569,636, filed Sep. 29, 2009, incorporated by reference herein, tohave higher stability in air as compared to the sensitivity in air ofthe E. coli [4Fe-4S] DHAD, which is desirable for obtaining betteractivity in a heterologous host cell.

Furthermore, as described herein, it has been found that expressing aheterologous DHAD protein at higher levels can provide increased DHADactivity when expressed in a host cell. High expression of a recombinantpolynucleotide can be accomplished in at least two ways: 1) byincreasing the copy number of a plasmid comprising the recombinantpolynucleotide; or 2) by integrating multiple copies of the gene ofinterest into the host cell's chromosome. As exemplified herein,expression of multiple copies of the heterologous DHAD, provides anincrease in specific activity of heterologous DHAD

Recombinant polynucleotides are typically cloned for expression usingthe coding sequence as part of a chimeric gene used for transformation,which includes a promoter operably linked to the coding sequence as wellas a ribosome binding site and a termination control region. The codingregion may be from the host cell for transformation and combined withregulatory sequences that are not native to the natural gene encodingDHAD. Alternatively, the coding region may be from another host cell.

Vectors useful for the transformation of a variety of host cells arecommon and described in the literature. Typically the vector contains aselectable marker and sequences allowing autonomous replication orchromosomal integration in the desired host. In addition, suitablevectors may comprise a promoter region which harbors transcriptionalinitiation controls and a transcriptional termination control region,between which a coding region DNA fragment may be inserted, to provideexpression of the inserted coding region. Both control regions may bederived from genes homologous to the transformed host cell, although itis to be understood that such control regions may also be derived fromgenes that are not native to the specific species chosen as a productionhost.

Yeast cells that can be hosts for expression or over-expression of aheterologous bacterial DHAD are any yeast cells that are amenable togenetic manipulation and include, but are not limited to, Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia,Issatchenkia, and Pichia. Suitable strains include, but are not limitedto, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces thermotolerans, Candida glabrata, Candidaalbicans, Pichia stipitis and Yarrowia lipolytica. In one embodiment,the host is Saccharomyces cerevisiae.

Expression is achieved by transforming a host cell with a genecomprising a sequence encoding DHAD, for example, a DHAD listed inTables 4a, 4b, 5 or 6, or identified using the screening methods inrelated U.S. application Ser. No. 12/569,636, filed Sep. 29, 2009,incorporated by reference herein. The coding region for the DHAD to beexpressed may be codon optimized for the target host cell, as well knownto one skilled in the art. Methods for gene expression in yeast areknown in the art (see, e.g., Methods in Enzymology, Volume 194, Guide toYeast Genetics and Molecular and Cell Biology (Part A, 2004, ChristineGuthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego,Calif.). Expression of genes in yeast typically requires a promoter,operably linked to a coding region of interest, and a transcriptionalterminator. A number of yeast promoters can be used in constructingexpression cassettes for genes in yeast, including, but not limited to,promoters derived from the following genes: CYC1, HIS3, GAL1, GAL10,ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA,GPD, GPM, and AOX1. Suitable transcriptional terminators include, butare not limited to, FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1.

Suitable promoters, transcriptional terminators, and DHAD coding regionsmay be cloned into E. coli-yeast shuttle vectors, and transformed intoyeast cells. These vectors allow strain propagation in both E. coli andyeast strains. In one embodiment, the vector used contains a selectablemarker and sequences allowing autonomous replication or chromosomalintegration in the desired host. Examples of plasmids used in yeast areshuttle vectors pRS423, pRS424, pRS425, and pRS426 (American TypeCulture Collection, Manassas, Va.), which contain an E. coli replicationorigin (e.g., pMB1), a yeast 2-micron origin of replication, and amarker for nutritional selection. The selection markers for these fourvectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vectorpRS425) and Ura3 (vector pRS426). Construction of expression vectorswith a chimeric gene encoding the described DHADs can be performed byeither standard molecular cloning techniques in E. coli or by the gaprepair recombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficienthomologous recombination in yeast. For example, a yeast vector DNA isdigested (e.g., in its multiple cloning site) to create a “gap” in itssequence. A number of insert DNAs of interest are generated that containa ≥21 bp sequence at both the 5′ and the 3′ ends that sequentiallyoverlap with each other, and with the 5′ and 3′ terminus of the vectorDNA. For example, to construct a yeast expression vector for “Gene X,” ayeast promoter and a yeast terminator are selected for the expressioncassette. The promoter and terminator are amplified from the yeastgenomic DNA, and Gene X is either PCR amplified from its source organismor obtained from a cloning vector comprising Gene X sequence. There isat least a 21 bp overlapping sequence between the 5′ end of thelinearized vector and the promoter sequence, between the promoter andGene X, between Gene X and the terminator sequence, and between theterminator and the 3′ end of the linearized vector. The “gapped” vectorand the insert DNAs are then co-transformed into a yeast strain andplated on the medium containing the appropriate compound mixtures thatallow complementation of the nutritional selection markers on theplasmids. The presence of correct insert combinations can be confirmedby PCR mapping using plasmid DNA prepared from the selected cells. Theplasmid DNA isolated from yeast (usually low in concentration) can thenbe transformed into an E. coli strain, e.g. TOP10, followed by minipreps and restriction mapping to further verify the plasmid construct.Finally, the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome alsotakes advantage of the homologous recombination system in yeast. Forexample, a cassette containing a coding region plus control elements(promoter and terminator) and auxotrophic marker is PCR-amplified with ahigh-fidelity DNA polymerase using primers that hybridize to thecassette and contain 40-70 base pairs of sequence homology to theregions 5′ and 3′ of the genomic area where insertion is desired. ThePCR product is then transformed into yeast and plated on mediumcontaining the appropriate compound mixtures that allow selection forthe integrated auxotrophic marker. For example, to integrate “Gene X”into chromosomal location “Y”, the promoter-coding regionX-terminatorconstruct is PCR amplified from a plasmid DNA construct and joined to anautotrophic marker (such as URA3) by either SOE PCR or by commonrestriction digests and cloning. The full cassette, containing thepromoter-coding regionX-terminator-URA3 region, is PCR amplified withprimer sequences that contain 40-70 bp of homology to the regions 5′ and3′ of location “Y” on the yeast chromosome. The PCR product istransformed into yeast and selected on growth media lacking uracil.Transformants can be verified either by colony PCR or by directsequencing of chromosomal DNA.

In addition to the above materials and methods that may be used toexpress a heterologous DHAD, these same, or similar, materials andmethods may be used to over-express a heterologous DHAD usingmodifications known to one of skill in the art. For example, when usinga plasmid-based system to over-express the recombinant polynucleotide, ahigh-copy number vector, or a vector with a copy number that can beregulated, may be constructed. Such a regulatable or inducible system isdescribed herein in Example 1; however, other systems are known to oneof skill in the art and may be used to construct other high-copy numberor copy number regulatable vectors. Alternatively, when using anintegration-based system to over-express the recombinant polypeptide, anintegration vector is required for targeting at multiple integrationsites. A multiple integration-based system is described herein inExample 2; however, other multiple integration-based systems are knownto one of skill in the art and may be used to target multipleintegrations of a recombinant polypeptide, for example integration intorDNA regions.

Expression of the heterologous DHAD in the recombinant host cell can bequantified, e.g., by a percent of total cell protein. Suchover-expression can be quantified in an amount selected from the groupconsisting of: (a) greater than about 0.001% of total cell protein; (b)greater than about 0.01% of total cell protein; (c) greater than about0.1% of total cell protein; (d) greater than about 0.5% of total cellprotein; (e) greater than about 1.0% of total cell protein; (f) greaterthan about 2.0% of total cell protein; (g) greater than about 5% oftotal cell protein; (h) greater than about 10% of total cell protein;and (i) greater than about 20% of total cell protein.

The specific activity of the heterologous DHAD produced in a recombinanthost cell can be quantified, e.g., as U/mg. The heterologous DHADspecific activity can be selected from the group consisting of: (a)greater than about 0.25 U/mg; (b) greater than about 0.3 U/mg; (c)greater than about 0.5 U/mg; (d) greater than about 1.0 U/mg; (e)greater than about 1.5 U/mg; (f) greater than about 2.0 U/mg; (g)greater than about 3.0 U/mg; (h) greater than about 4.0 U/mg; (i)greater than about 5.0 U/mg; (j) greater than about 6.0 U/mg; (k)greater than about 7.0 U/mg; (l) greater than about 8.0 U/mg; (m)greater than about 9.0 U/mg; (n) greater than about 10.0 U/mg; (o)greater than about 20.0 U/mg; and (p) greater than about 50.0 U/mg.

The heterologous DHAD specific activity can also be quantified, e.g., asa percent comparison to an endogenous DHAD specific activity or to someother control DHAD specific activity. An example of a “control” DHADspecific activity is that from a heterologous DHAD expressed in arecombinant host cell using a low copy number plasmid or a plasmid thatis not otherwise inducible or regulatable. Such a control establishes abaseline from which to compare the specific activity of the sameheterologous DHAD expressed in a recombinant host cell using a high copynumber plasmid or a plasmid with copy number that can be regulated, orco-expressed with polynucleotides encoding polypeptides affecting Fe—Scluster biosynthesis or Fe uptake and utilization, as described below.Thus, the increase in specific activity of the heterologous DHAD whencompared to the control DHAD specific activity can be in an amountselected from the group consisting of: greater than an about 10%increase; greater than an about 20% increase; greater than an about 30%increase; greater than an about 40% increase; greater than an about 50%increase; greater than an about 60% increase; greater than an about 70%increase; greater than an about 80% increase; greater than an about 90%increase; greater than an about 95% increase; greater than an about 98%increase; and greater than an about 99% increase. The heterologous DHADspecific activity can also be expressed by “fold increase” over control.Thus, the increase in specific activity can be selected from the groupconsisting of: (a) greater than about 2-fold higher, (b) greater thanabout 5-fold higher, (c) greater than about 8-fold higher, or (d)greater than about 10-fold higher than control.

Fe—S Cluster Forming Proteins and Fe Regulation, Utilization, andHomeostasis

As described above, DHAD enzymes require Fe—S clusters for functioning,therefore, they must be expressed in a host having the genetic machineryto produce and load Fe—S clusters into the apo-protein if they are goingto be expressed in functional form. As described elsewhere herein, innormal yeast, the mitochondria play an important role in Fe—S clusterbiosynthesis. The flux in the formation and movement of Fe—S clusterprecursors from mitochondria to Fe—S cluster requiring proteins in thecytosol of normal yeast is believed to be limited. For example, after apoint a further increase in the expression of the protein ofheterologous DHADs in the cytosol does not result in a correspondingincrease in DHAD activity. While not wishing to be bound by theory, itis believed that this is because the increased amounts of theheterologous DHAD are not getting loaded with the Fe—S cluster requisitefor activity because the cell is not able to supply the increased demandfor Fe—S clusters that arises in the conditions described above.Demonstrated herein is that yeast cells can be genetically modified in 2ways (separately or contemporaneously) that will result in an increasedfraction of the heterologous DHAD expressed in the cytosol being loadedwith its requisite Fe—S cluster. One way is to modify the expression ofyeast genes involved in the Fe—S cluster formation, such as Fe—S clusterbiosynthesis pathway genes or Fe uptake and utilization genes. The otherway is to express heterologous genes involved in Fe—S clusterbiosynthesis or Fe uptake and utilization in the cytoplasm of yeast.

Yeast genes that encode polypeptides that are involved in Fe uptake andutilization and Fe—S cluster biosynthesis are candidates formodification of expression. In embodiments, the modification results inincreased function of a selected Fe—S cluster requiring protein.

As an example, Aft1 has been found to act as a transcriptional activatorfor genes into the iron regulon (Kumanovics, et al. J. Biol. Chem.,2008. 283, p. 10276-10286; Li, H., et al., The Yeast Iron RegulatoryProteins Grx3/4 and Fra2 form Heterodimeric Complexes Containing a[2Fe-2S] Cluster with Cysteinyl and Histidyl Ligation. Biochemistry,2009. 48(40): p. 9569-9581. As exemplified herein, the deletion of knowninhibitors of Aft1 translocation, results in an increase in specificactivity of an Fe—S cluster requiring protein because it leads to anincrease Fe—S cluster loading of the protein. While not wishing to bebound by theory, it is thus believed that altering expression of certaingenes of the Fe regulon, whether directly or through deletion orupregulation of inhibitors, will likewise increase the loading andfunction of Fe—S cluster requiring proteins. For example, genes thatplay a role in, or are part of, Fe utilization and homeostasis in yeast,such as Fe Regulon genes, may be targeted for altered expression. Suchgenes are known in the art, and examples of these genes are listed inTable 7. (The list in Table 7 is taken from Rutherford, J. C., et al.,Activation of the Iron Regulon by the Yeast Aft1/Aft2 TranscriptionFactors Depends on Mitochondrial but Not Cytosolic Iron-Sulfur ProteinBiogenesis., J. Biol. Chem., 2005. 280(11): p. 10135-10140; Foury, F.and D. Talibi, Mitochondrial control of iron homeostasis. A genome wideanalysis of gene expression in a yeast frataxin-deficient strain. J.Biol. Chem., 2001. 276(11): p. 7762-7768; and Shakoury-Elizeh, M., etal., Transcriptional remodeling in response to iron deprivation inSaccharomyces cerevisiae. Mol. Biol. Cell, 2004. 15(3): p. 1233-1243.)

TABLE 7 Examples of yeast genes associated with Fe uptake andutilization. Nucleic Amino Acid Acid Gene SEQ ID SEQ Name PutativeFunction NO: ID NO: ARN1 Transporter, member of the ARN family oftransporters that 805 738 specifically recognize siderophore-ironchelates; responsible for uptake of iron bound to ferrirubin,ferrirhodin, and related siderophores ARN2 Transporter, member of theARN family of transporters that 806 739 specifically recognizesiderophore-iron chelates; responsible for uptake of iron bound to thesiderophore triacetylfusarinine C ATX1 Cytosolic copper metallochaperonethat transports copper to 802 735 the secretory vesicle coppertransporter Ccc2p for eventual insertion into Fet3p, which is amulticopper oxidase required for high-affinity iron uptake CCC2Cu(+2)-transporting P-type ATPase, required for export of 803 736 copperfrom the cytosol into an extracytosolic compartment; has similarity tohuman proteins involved in Menkes and Wilsons diseases COT1 Vacuolartransporter that mediates zinc transport into the 816 749 vacuole;overexpression confers resistance to cobalt and rhodium ENB1 Endosomalferric enterobactin transporter, expressed under 808 741 (ARN4)conditions of iron deprivation; member of the major facilitatorsuperfamily; expression is regulated by Rcs1p and affected bychloroquine treatment FET3 Ferro-O2-oxidoreductase required forhigh-affinity iron 800 733 uptake and involved in mediating resistanceto copper ion toxicity, belongs to class of integral membranemulticopper oxidases FET5 Multicopper oxidase, integral membrane proteinwith 814 747 similarity to Fet3p; may have a role in iron transport FIT1Mannoprotein that is incorporated into the cell wall via a 792 725glycosylphosphatidylinositol (GPI) anchor, involved in the retention ofsiderophore-iron in the cell wall FIT2 Mannoprotein that is incorporatedinto the cell wall via a 793 726 glycosylphosphatidylinositol (GPI)anchor, involved in the retention of siderophore-iron in the cell wallFIT3 Mannoprotein that is incorporated into the cell wall via a 794 727glycosylphosphatidylinositol (GPI) anchor, involved in the retention ofsiderophore-iron in the cell wall FRE1 Ferric reductase and cupricreductase, reduces siderophore- 795 728 bound iron and oxidized copperprior to uptake by transporters; expression induced by low copper andiron levels FRE2 Ferric reductase and cupric reductase, reducessiderophore- 796 729 bound iron and oxidized copper prior to uptake bytransporters; expression induced by low copper and iron levels FRE3Ferric reductase, reduces siderophore-bound iron prior to 797 730 uptakeby transporters; expression induced by low iron levels FRE4 Ferricreductase, reduces a specific subset of siderophore- 798 731 bound ironprior to uptake by transporters; expression induced by low iron levelsFRE5 Putative ferric reductase with similarity to Fre2p; expression 799732 induced by low iron levels; the authentic, non-tagged protein isdetected in highly purified mitochondria in high- throughput studiesFRE6 Putative ferric reductase with similarity to Fre2p; expression 817750 induced by low iron levels FTH1 Putative high affinity irontransporter involved in transport of 813 746 intravacuolar stores ofiron; forms complex with Fet5p; expression is regulated by iron;proposed to play indirect role in endocytosis FTR1 High affinity ironpermease involved in the transport of iron 801 734 across the plasmamembrane; forms complex with Fet3p; expression is regulated by iron HMX1ER localized, heme-binding peroxidase involved in the 823 756degradation of heme; does not exhibit heme oxygenase activity despitesimilarity to heme oxygenases; expression regulated by AFT1 SIT1Ferrioxamine B transporter, member of the ARN family of 807 740 (ARN3)transporters that specifically recognize siderophore-iron chelates;transcription is induced during iron deprivation and diauxic shift;potentially phosphorylated by Cdc28p SMF3 Putative divalent metal iontransporter involved in iron 815 741 homeostasis; transcriptionallyregulated by metal ions; member of the Nramp family of metal transportproteins TIS11 mRNA-binding protein expressed during iron starvation;824 757 (CTH2) binds to a sequence element in the 3′-untranslatedregions of specific mRNAs to mediate their degradation; involved in ironhomeostasis VHT1 High-affinity plasma membrane H+-biotin (vitamin H) 822755 symporter; mutation results in fatty acid auxotrophy; 12transmembrane domain containing major facilitator subfamily member; mRNAlevels negatively regulated by iron deprivation and biotin

Based on their functions and association with Fe uptake and utilization,the proteins encoded by the genes disclosed in Table 7 are candidatesfor affecting Fe—S cluster biosynthesis. Additional yeast genesassociated with Fe uptake and utilization or Fe—S cluster biosynthesisinclude those listed in Table 8.

TABLE 8 Genes Associated With Yeast Fe Uptake and Utilization or Fe—SCluster Biosynthesis Nucleic Amino Acid Acid Gene SEQ ID SEQ ID Name NO:NO: Putative Function AFT1 770 703 Transcription factor involved in ironutilization and homeostasis; binds the consensus site PyPuCACCCPu andactivates the expression of target genes in response to changes in ironavailability AFT2 771 704 Iron-regulated transcriptional activator;activates genes involved in intracellular iron use and required for ironhomeostasis and resistance to oxidative stress; similar to Aft1p AIM1779 712 Interacts with Grx3/4 ARH1 855 837 Oxidoreductase of themitochondrial inner membrane, involved in cytoplasmic and mitochondrialiron homeostasis and required for activity of Fe—S cluster-containingenzymes; one of the few mitochondrial proteins essential for viability(see, e.g., Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700(2008)) ATM1 830 763 Mitochondrial inner membrane ATP-binding cassette(ABC) transporter, exports mitochondrially synthesized precursors ofiron-sulfur (Fe/S) clusters to the cytosol (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) BUD32 778 711Interacts with Grx3/4 and Aft1p CAD1 791 724 Stress responses includingFe deprivation; also regulates CTI6 and MRS4 (YAP2) genes CCC1 811 744Putative vacuolar Fe2+/Mn2+ transporter; suppresses respiratory deficitof yfh1 mutants, which lack the ortholog of mammalian frataxin, bypreventing mitochondrial iron accumulation CFD1 834 767 Highlyconserved, iron-sulfur cluster binding protein localized in thecytoplasm; forms a complex with Nbp35p that is involved in iron-sulfurprotein assembly in the cytosol (see, e.g., Lill, R. and U. Muehlenhoff,Ann. Rev. Biochem. 77: 669-700 (2008)) CIA1 836 769 WD40 repeat proteininvolved in assembly of cytosolic and nuclear iron- sulfur proteins;similar to the human Ciaol protein; YDR267C is an essential gene (see,e.g., Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700(2008)) CMK1 784 717 Interacts with Grx4p CTH1 825 758 mRNA binding anddegradation under Fe depletion conditions CTI6 786 719 Growth in lowiron conditions CYC8 787 720 General transcriptional co-repressor, actstogether with Tup1p; also acts as (SSN6) part of a transcriptionalco-activator complex that recruits the SWI/SNF and SAGA complexes topromoters; can form the prion [OCT+] DAP1 820 753 DRE2 781 714 Interactswith Grx3p ERV1 856 838 Flavin-linked sulfhydryl oxidase of themitochondrial intermembrane space (IMS), oxidizes Mia40p as part of adisulfide relay system that promotes IMS retention of imported proteins;ortholog of human hepatopoietin (ALR) (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) Central players ofthe export pathway are the ABC transporter Atm1p of the mitochondrialinner membrane, the sulfhydryl oxidase Erv1p of the intermembrane space,and the tripeptide glutathione (23, 27, 50) (see Gerber, J., et al.,Mol. Cell. Biol. 24(11): 4848-57 (2004)) ESA1 782 715 Interacts withGrx4p/Aft1p FET4 809 742 Low-affinity Fe(II) transporter of the plasmamembrane FRA1 772 705 Protein involved in negative regulation oftranscription of iron regulon; forms an iron independent complex withFra2p, Grx3p, and Grx4p; cytosolic; mutant fails to represstranscription of iron regulon and is defective in spore formation FRA2773 706 Protein involved in negative regulation of transcription of ironregulon; forms an iron independent complex with Fra2p, Grx3p, and Grx4p;null mutant fails to repress iron regulon and is sensitive to nickelGEF1 804 737 Copper transporter/loading for Fet3p GGC1 857 839Mitochondrial GTP/GDP transporter, essential for mitochondrial genome(YHM1) maintenance; has a role in mitochondrial iron transport; memberof the mitochondrial carrier family GRX1 858 840 Hydroperoxide andsuperoxide-radical responsive heat-stable glutathione- dependentdisulfide oxidoreductase with active site cysteine pair; protects cellsfrom oxidative damage GRX2 832 765 Cytoplasmic glutaredoxin,thioltransferase, glutathione-dependent disulfide oxidoreductaseinvolved in maintaining redox state of target proteins, also exhibitsglutathione peroxidase activity, expression induced in response tostress GRX3 774 707 Hydroperoxide and superoxide-radical responsiveglutathione-dependent oxidoreductase; monothiol glutaredoxin subfamilymember along with Grx4p and Grx5p; protects cells from oxidative damageGRX4 775 708 Hydroperoxide and superoxide-radical responsiveglutathione-dependent oxidoreductase; monothiol glutaredoxin subfamilymember along with Grx3p and Grx5p; protects cells from oxidative damage.GRX5 831 764 Hydroperoxide and superoxide-radical responsiveglutathione-dependent oxidoreductase; mitochondrial matrix proteininvolved in the synthesis/assembly of iron-sulfur centers; monothiolglutaredoxin subfamily member along with Grx3p and Grx4p (see, e.g.,Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) HDA1790 723 Interacts with Tup1p, Ssn6p for Aft1/2p regulation in theabsence of heme IBA57 859 841 Mitochondrial matrix protein involved inthe incorporation of iron-sulfur clusters into mitochondrialaconitase-type proteins; activates the radical-SAM family members Bio2pand Lip5p; interacts with Ccr4p in the two-hybrid system (see, e.g.,Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) ISA1860 842 Mitochondrial matrix protein involved in biogenesis of theiron-sulfur (Fe/S) cluster of Fe/S proteins, isa1 deletion causes lossof mitochondrial DNA and respiratory deficiency; depletion reducesgrowth on nonfermentable carbon sources (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) ISA2 861 843 Proteinrequired for maturation of mitochondrial and cytosolic Fe/S proteins,localizes to the mitochondrial intermembrane space, overexpression ofISA2 suppresses grx5 mutations (see, e.g., Lill, R. and U. Muehlenhoff,Ann. Rev. Biochem. 77: 669-700 (2008)) ISU1 828 761 Conserved protein ofthe mitochondrial matrix, performs a scaffolding function duringassembly of iron-sulfur clusters, interacts physically and functionallywith yeast frataxin (Yfh1p); isu1 isu2 double mutant is inviable (see,e.g., Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700(2008)) ISU2 829 762 Conserved protein of the mitochondrial matrix,required for synthesis of mitochondrial and cytosolic iron-sulfurproteins, performs a scaffolding function in mitochondria during Fe/Scluster assembly; isu1 isu2 double mutant is inviable (see, e.g., Lill,R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) JAC1 862844 Specialized J-protein that functions with Hsp70 in Fe—S clusterbiogenesis in mitochondria, involved in iron utilization; contains a Jdomain typical to J- type chaperones; localizes to the mitochondrialmatrix (see, e.g., Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77:669-700 (2008)) MGE1 863 845 Mitochondrial matrix cochaperone, acts as anucleotide release factor for Ssc1p in protein translocation andfolding; also acts as cochaperone for Ssq1p in folding of Fe—S clusterproteins; homolog of E. coli GrpE (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) MRS3 819 752 Irontransporter that mediates Fe2+ transport across the inner mitochondrialmembrane; mitochondrial carrier family member, similar to andfunctionally redundant with Mrs4p; active under low-iron conditions; maytransport other cations (see, e.g., Lill, R. and U. Muehlenhoff, Ann.Rev. Biochem. 77: 669-700 (2008)) MRS4 818 751 Iron transporter thatmediates Fe2+ transport across the inner mitochondrial membrane;mitochondrial carrier family member, similar to and functionallyredundant with Mrs3p; active under low-iron conditions; may transportother cations (see, e.g., Lill, R. and U. Muehlenhoff, Ann. Rev.Biochem. 77: 669-700 (2008)) MSN5 776 709 Exporting Aft1p and otherproteins from the nucleus NAR1 833 766 Component of the cytosoliciron-sulfur (FeS) protein assembly machinery, required for maturation ofcytosolic and nuclear FeS proteins and for normal resistance tooxidative stress; homologous to human Narf (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) NBP35 835 768Essential iron-sulfur cluster binding protein localized in thecytoplasm; forms a complex with Cfd1p that is involved in iron-sulfurprotein assembly in the cytosol; similar to P-loop NTPases (see, e.g.,Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) NFS1864 846 Cysteine desulfurase involved in iron-sulfur cluster (Fe/S)biogenesis; required for the post-transcriptional thio-modification ofmitochondrial and cytoplasmic tRNAs; essential protein locatedpredominantly in mitochondria (see, e.g., Lill, R. and U. Muehlenhoff,Ann. Rev. Biochem. 77: 669-700 (2008)) NFU1 865 847 Protein involved iniron utilization in mitochondria; similar to NifU, which is a proteinrequired for the maturation of the Fe/S clusters of nitrogenase innitrogen-fixing bacteria (see, e.g., Lill, R. and U. Muehlenhoff, Ann.Rev. Biochem. 77: 669-700 (2008)) NHP6a 788, 789 721, 722 Both arehigh-mobility group non-histone chromatin protein, functionally and bredundant with Nhp6Bp; homologous to mammalian high mobility groupproteins 1 and 2; acts to recruit transcription factor Rcs1p to certainpromoters PSE1 777 710 Importing Aft1p and other proteins to the nucleusSMF1 810 743 Low affinity Fe(II) transporter of the plasma membrane SNF1866 848 AMP-activated serine/threonine protein kinase found in a complexcontaining Snf4p and members of the Sip1p/Sip2p/Gal83p family; requiredfor transcription of glucose-repressed genes, thermotolerance,sporulation, and peroxisome biogenesis SNF2 867 849 Catalytic subunit ofthe SWI/SNF chromatin remodeling complex involved in transcriptionalregulation; contains DNA-stimulated ATPase activity; functionsinterdependently in transcriptional activation with Snf5p and Snf6p SNF3868 850 Plasma membrane glucose sensor that regulates glucose transport;has 12 predicted transmembrane segments; long cytoplasmic C-terminaltail is required for low glucose induction of hexose transporter genesHXT2 and HXT4 SNF4 869 851 Activating gamma subunit of the AMP-activatedSnf1p kinase complex (contains Snf1p and a Sip1p/Sip2p/Gal83p familymember); activates glucose-repressed genes, represses glucose-inducedgenes; role in sporulation, and peroxisome biogenesis SSQ1 827 760Mitochondrial hsp70-type molecular chaperone, required for assembly ofiron/sulfur clusters into proteins at a step after cluster synthesis,and for maturation of Yfh1p, which is a homolog of human frataxinimplicated in Friedreich's ataxia (see, e.g., Lill, R. and U.Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) TIM12 871 853Essential protein of the inner mitochondrial membrane, peripherally(MRS5) localized; component of the TIM22 complex, which is a twin-poretranslocase that mediates insertion of numerous multispanning innermembrane protein. TUP1 785 718 General repressor of transcriptionNP_011911.1 821 754 VPS41 872 854 Vacuolar membrane protein that is asubunit of the homotypic vacuole fusion (FET2) and vacuole proteinsorting (HOPS) complex; essential for membrane docking and fusion at theGolgi-to-endosome and endosome-to-vacuole stages of protein transportYAH1 870 852 Ferredoxin of the mitochondrial matrix required forformation of cellular iron-sulfur proteins; involved in heme Abiosynthesis; homologous to human adrenodoxin (see, e.g., Lill, R. andU. Muehlenhoff, Ann. Rev. Biochem. 77: 669-700 (2008)) YAP5 812 745Regulation (CCC1) YFH1 826 759 Mitochondrial matrix iron chaperone;oxidizes and stores iron; interacts with (Frataxin) Isu1p to promoteFe—S cluster assembly; mutation results in multiple Fe/S- dependentenzyme deficiencies; human frataxin homolog is mutated in Friedrich'sataxia (see, e.g., Lill, R. and U. Muehlenhoff, Ann. Rev. Biochem. 77:669-700 (2008)) YRA1 783 716 Interacts with Grx4p ZPR1 780 713 Interactswith Aft1p

Additional genes encoding polypeptides affecting Fe—S clusterbiosynthesis from other host cells have been identified and include, butare not limited to, those genes listed in Table 9.

TABLE 9 Genes Directly Involved in Fe—S Cluster Biosynthesis fromVarious Cells Gene Name SEQ ID NOs(Amino Acid, Nucleic Function Acid)(Accession; CDS) Azotobacter vinelandii nif genes (FIGS. 6A and 6B; seeJohnson, D. C., et al., Ann. Rev. Biochem. 74: 247-81 (2005)) iscA^(nif)[Fe—S] cluster scaffold protein (see Johnson, D. C., et al., Ann. Rev.(873, 894) Biochem. 74: 247-81 (2005)) (YP_002797399.1; nucleotides153037 to 153360 of NC_012560.1) nifU NifU is a scaffold protein forassembly and transfer of iron-sulfur (875, 896) clusters (see Johnson,D. C., et al., Ann. Rev. Biochem. 74: 247-81 (2005)). (YP_002797400.1;nucleotides 153425 to 154363 of NC_012560.1) nifS Cysteine desulfuraseinvolved in the mobilization of S for nitrogenase (874, 895) maturation(see Johnson, D. C., et al., Ann. Rev. Biochem. 74: 247-81 (2005)).(YP_002797401.1; nucleotides 154365 to 155573 of NC_012560.1) cysE1Involved in cysteine biosynthesis (see Johnson, D. C., et al., Ann. Rev.(876, 897) Biochem. 74: 247-81 (2005)) (YP_002797403.1; nucleotides156797 to 157594 of NC_012560.1) cysE2 Involved in cysteine biosynthesis(see Johnson, D. C., et al., Ann. Rev. (929, 947) Biochem. 74: 247-81(2005)) (YP_002801153.1; reverse complement of nucleotides 4092159 to4092938 of NC_012560.1) iscS Cysteine desulfurase involved in themobilization of S (see Johnson, D. C., (930, 948) et al., Ann. Rev.Biochem. 74: 247-81 (2005)) (YP_002801151.1; reverse complement ofnucleotides of 4090290 to 4091504 of NC_012560.1) iscU [Fe—S] clusterscaffold protein (see Johnson, D. C., et al., Ann. Rev. (931, 949)Biochem. 74: 247-81 (2005)) (YP_002801150.1; reverse complement ofnucleotides 4089860 to 4090246 of NC_012560.1) iscA [Fe—S] clusterscaffold protein (see Johnson, D. C., et al., Ann. Rev. (932, 950)Biochem. 74: 247-81 (2005)) (YP_002801149.1; reverse complement ofnucleotides 4089511 to 4089834 of NC_012560.1) hscB HscB heat shockcognate protein associated with Isc-directed [Fe—S] (933, 951) proteinmaturation (see Johnson, D. C., et al., Ann. Rev. Biochem. 74: 247-81(2005)) (YP_002801148.1; reverse complement of nucleotides 4088980 to4089501 of NC_012560.1) hscA HscA heat shock cognate protein associatedwith Isc-directed [Fe—S] (934, 952) protein maturation (see Johnson, D.C., et al., Ann. Rev. Biochem. 74: 247-81 (2005)) (YP_002801147.1;reverse complement of nucleotides 4087072 to 4088937 of NC_012560.1) FdxFerredoxin (935, 953) (YP_002801146.1; reverse complement of nucleotides4086730 to 4087071 of NC_012560.1) sufS Cysteine desulfurase involved inthe mobilization of S (see Johnson, D. C., (936, 954) et al., Ann. Rev.Biochem. 74: 247-81 (2005)) (YP_002801025.1; nucleotides 3961166 to3962515 of NC_012560.1) sufE (YP_002801026.1; nucleotides 3962512 to3962916 of NC_012560.1) (937, 955) cysE3 Involved in cysteinebiosynthesis (see Johnson, D. C., et al., Ann. Rev. (938, 956) Biochem.74: 247-81 (2005)) (YP_002799274.1; nucleotides 2093069 to 2094052 ofNC_012560.1) sufS2 Cysteine desulfurase involved in the mobilization ofS (see Johnson, D. C., (939, 957) et al., Ann. Rev. Biochem. 74: 247-81(2005)) (YP_002799276.1; nucleotides 2095267 to 2097081 of NC_012560.1)iscA2 also [Fe—S] cluster scaffold protein (see Johnson, D. C., et al.,Ann. Rev. known as Biochem. 74: 247-81 (2005)) eprA (YP_002801687.1;reverse complement of nucleotides 4681573 to (877, 898) 4681923 ofNC_012560.1) Nfu also Human nfu appears to be a persulfide reductaseaccording to the known as equation shown in FIG. 6C. (see Liu, Y., W.Qi, and J. A. Cowan, NfuA Biochem. 48(5): 973-80 (2009)) (878, 899)(YP_002800022.1; reverse complement of nucleotides 2961161 to 2961745 ofNC_012560.1) nfuA also Spectroscopic and analytical studies indicatethat one known as [4Fe—4S] cluster can be assembled in vitro within adimeric form AnfU of NfuA. The resultant [4Fe—4S] cluster-loaded form ofNfuA is (879, 900) competent for rapid in vitro activation ofapo-aconitase. Based on these results a model is proposed where NfuAcould represent a class of intermediate [Fe—S] cluster carriers involvedin [Fe—S] protein maturation. (see Bandyopadhyay, S., et al., J Biol.Chem. 283(20): 14092-99 (2008)) (YP_002801977.1; nucleotides 4963727 to4964017 of NC_012560.1) nfuV also Could have specialized functionsrelated to the maturation, protection, known as or repair of specific[Fe—S] proteins (see Johnson, D. C., et al., Ann. Rev. VnfU Biochem. 74:247-81 (2005)). (880, 901) (YP_002797514.1; reverse complement ofnucleotides 263828 to 264118 of NC_012560.1) Helicobacter pylori nifgenes (FIG. 7; see Johnson, D. C., et al., Ann. Rev. Biochem. 74: 247-81(2005)) nifS NifS is a cysteine desulfurase. (881, 902) (YP_003057033.1;nucleotides 218891 to 220054 of NC_012973.1) nifU NifU is a scaffoldprotein for assembly and transfer of iron-sulfur (882, 903) clusters.(YP_003057034.1; nucleotides 220076 to 221056 of NC_012973.1) nfu(YP_003058109.1; nucleotides 1448886 to 1449155 of NC_012973.1) (927,945) iscS (YP_003057709.1; reverse complement of nucleotides 1012615 to(928, 946) 1013937 of NC_012973.1) E. coli isc genes (FIG. 8; seeJohnson, D. C., et al., Ann. Rev. Biochem. 74: 247-81 (2005)) iscSEcoCyc: IscS is a cysteine desulfurase that catalyzes the conversion of(883, 904) cysteine into alanine and sulfur via intermediate formationof a cysteine persulfide. (YP_026169.1; reverse complement ofnucleotides 2658339 to 2659553 of NC_000913.2) iscU EcoCyc: IscU is ascaffold protein for assembly and transfer of iron- (884, 905) sulfurclusters. IscU is able to form 2Fe—2S clusters and transfer them toapo-ferredoxin, acting catalytically. The chaperones HscA and HscB andATP hydrolysis by HscA accelerate cluster transfer. (NP_417024.1;reverse complement of nucleotides 2657925 to 2658311 of NC_000913.2)iscA EcoCyc: IscA is an iron-sulfur cluster assembly protein that formsthe (885, 906) [2Fe—2S] cluster of ferredoxin. It has been shown to bindiron with an apparent association constant of 3 × 10−19M⁻¹. In vitro inthe presence of IscS and cysteine, IscA can provide iron to iscU. Native[2Fe—2S] SufA can transfer its Fe—S cluster to both [2Fe—2S] and[4Fe—4S] apoproteins. (see Gupta, V., et al., J. Am. Chem. Soc. 131(17):6149-53 (2009)) The results suggest that the biogenesis of the [4Fe—4S]clusters and the [2Fe—2S] clusters may have distinct pathways and thatIscA/SufA paralogues are essential for the [4Fe—4S] cluster assembly,but are dispensable for the [2Fe—2S] cluster assembly in E. coli underaerobic conditions. (Tan, G., et al., Biochem. J., 420(3): 463-72(2009)) (NP_417023.1; reverse complement of nucleotides 2657585 to2657908 of NC_000913.2) hscB EcoCyc: HscB is a co-chaperone thatstimulates HscA (Hsc66) ATPase (886, 907) activity. HscB does notexhibit its own chaperone activity. HscB is required for wild-typestimulation of HscA ATPase activity by the substrate, IscU, and forwild-type interaction between HscA and IscU. This system is involved iniron-sulfur cluster assembly. (NP_417022.1; reverse complement ofnucleotides 2656974 to 2657489 of NC_000913.2) hscA EcoCyc: Hsc66together with Hsc20 may comprise a chaperone system (887, 908) similarto DnaK/DnaJ. Hsc66 is required for the assembly of iron-sulfurclusters. IscU may be a substrate for Hsc66. In the presence of Hsc20,IscU stimulates the ATPase activity of Hsc66 up to 480-fold; the in vivoturnover rate of the chaperone cycle may be determined by theavailability of the IscU-Hsc20 complex. Hsc66 directly interacts withIscU, IscA, and Fdx. (NP_417021.1; reverse complement of nucleotides2655107 to 2656957 of NC_000913.2) Fdx EcoCyc: [2Fe—2S] ferridoxin (888,909) (NP_417020.1; reverse complement of nucleotides 2654770 to 2655105of NC_000913.2) E. coli suf genes (FIG. 9; see Johnson, D. C., et al.,Ann. Rev. Biochem. 74: 247-81 (2005)) sufA EcoCyc: SufA is part of theprotein machinery that is involved in the (889, 910) biosynthesis ofiron-sulfur clusters. In vitro, purified apoSufA can chelate iron-sulfurclusters by treatment with iron and sulfide under anaerobic conditions.HoloSufA then can form a fast and tight association with the targetapoprotein biotin synthase (BioB) and transfers a [4Fe—4S] cluster toBioB in a slow reaction. (NP_416199.1; reverse complement of nucleotides1762042 to 1762410 of NC_000913.2) sufB EcoCyc: The SufB-SufC-SufDcomplex activates the cysteine (890, 911) desulfurase activity SufS inconjunction with the SufE sulfur acceptor protein. (NP_416198.2; reversecomplement of nucleotides 1760546 to 1762033 of NC_000913.2) sufCEcoCyc: SufC is part of the protein machinery that is involved in the(891, 912) biosynthesis of iron-sulfur clusters. The SufB-SufC-SufDcomplex activates the cysteine desulfurase activity of SufS inconjunction with the SufE sulfur acceptor protein. (NP_416197.1; reversecomplement of nucleotides 1759790 to 1760536 of NC_000913.2) sufDEcoCyc: The SufB-SufC-SufD complex activates the cysteine (892, 913)desulfurase activity SufS in conjunction with the SufE sulfur acceptorprotein (NP_416196.1; reverse complement of nucleotides 1758544 to1759815 of NC_000913.2) sufS EcoCyc: SufS is a member of the NifSprotein family. SufS exhibits (893, 914) activity with respect toassembly of the ferredoxin iron-sulfur cluster in an in vitro assay.(NP_416195.1; reverse complement of nucleotides 1757327 to 1758547 ofNC_000913.2) sufE1 also (NP_416194.1; reverse complement of nucleotides1756898 to 1757314 known as suf E of NC_000913.2) (925, 943) sufS2 also(NP_417290.1; NC_000913.2 nucleotides 2941359 to 2942564) known as csdA(924, 942) sufE2 also (NP_417291.1; nucleotides 2942564 to 2943007 ofNC_000913.2) known as csdE (926, 944) iscA2 also (NP_414698.1;nucleotides 176610 to 176954 of NC_000913.2) known as erpA (922, 940)nfu also known (NP_417873.1; nucleotides 3543646 to 3544221 ofNC_000913.2) as nfuA (923, 941)

Fe uptake and metabolism and/or Fe—S cluster biosynthesis genes,including, but not limited to, those listed in Tables 7, 8 or 9 canpotentially be deleted, mutated, expressed, up-regulated, ordown-regulated to increase the flux in an Fe—S cluster biosynthesispathway and improve specific activity of Fe—S cluster requiring proteinssuch as DHAD. In addition, co-factors can be added to change theactivity of polypeptides having Fe—S cluster regulatory activity toincrease the flux in an Fe—S cluster biosynthesis pathway and improveDHAD specific activity.

For example, the genes that increase the flux in an Fe—S clusterbiosynthesis pathway can be expressed to improve the activity of DHAD byproviding an adequate amount of Fe—S clusters for the apo-enzyme. Anygene, or a combination of them, such as one or more genes listed inTables 7, 8, or 9, can be cloned and expressed in a pRS411 plasmid asdescribed in Example 4. The resulting constructs, along with the DHADexpression vector pHR81 FBA ilvD(Sm), can then be transformed intowild-type BY4741. As a control, pRS411 without any gene of interest andvector pHR81 FBA ilvD(Sm) are transformed into a wild-type strain. Thetransformants are selected on agar plates with SD medium without uraciland methionine to maintain both plasmids as described in Example 4.Enzymatic activity for DHAD in the crude extract of different strainsfrom the transformation can be measured. The results can be comparedwith the specific activity obtained from the control pRS411 without anygene of interest and vector pHR81 FBA ilvD(Sm) transformed into awild-type strain. An increase in specific activity indicates a gene thatcan be used to increase the flux in an Fe—S cluster biosynthesispathway.

In addition, strains with deletions in more than one of the genesinvolved in Fe—S cluster regulatory activity can be created to provideadditive effects in improving the enzymes or proteins containing Fe—Scluster(s). For example, double mutants with deletions in both FRA2 andGXR3 genes can be used to transform vector pHR81 FBA-IlvD(sm), and theDHAD activity in the crude extract from the transformants can bemeasured.

Another alternative is to alter the expression of, e.g., the PSE1 (SEQID NO:777) gene, which encodes a protein involved in the import of Aft1pinto the nucleus (Fukunaka, et al, 2003, J. Biological Chem., vol. 278,pp. 50120-50127). Expression of this gene can be accomplished by cloningit in vector pRS411 as described above.

Thus, provided herein are recombinant host cells that comprise analteration in the expression of any polypeptide encoded by an Fe uptakeand utilization or an Fe—S cluster biosynthesis gene. Encompassed arerecombinant host cells that comprise at least one heterologouspolynucleotide of any one of the above-referenced Fe—S clusterbiosynthesis genes. Also encompassed are recombinant host cells, whereinthe host cell comprises at least one deletion, mutation, and/orsubstitution in an endogenous gene of any one of the above-referenced Feuptake and utilization or Fe—S cluster biosynthesis genes. Also providedare recombinant host cells that comprise at least one heterologouspolynucleotide of any one of the above-referenced Fe uptake andutilization or Fe—S cluster biosynthesis genes, wherein the host cellcomprises at least one deletion, mutation, and/or substitution in anendogenous gene of any one of the above-referenced Fe uptake andutilization or Fe—S cluster biosynthesis genes.

These recombinant host cells can also comprise at least one heterologousFe—S cluster requiring protein. For example, provided herein is arecombinant host cell comprising at least one heterologous DHAD and atleast one heterologous polynucleotide encoding a polypeptide affectingFe—S cluster biosynthesis. Also provided is a recombinant host cellcomprising at least one heterologous DHAD, wherein the host cellcomprises at least one deletion, mutation, and/or substitution in anendogenous gene encoding a polypeptide affecting Fe—S clusterbiosynthesis. Also provided is a recombinant host cell comprising atleast one heterologous DHAD and at least one heterologous polynucleotideencoding a polypeptide affecting Fe—S cluster biosynthesis, wherein thehost cell comprises at least one deletion, mutation, and/or substitutionin an endogenous gene encoding a polypeptide affecting Fe—S clusterbiosynthesis.

Host cells that can be used in the present invention include yeast hostcells including, but not limited to, Saccharomyces, Schizosaccharomyces,Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, and Pichia.Bacterial host cells can also be used to create recombinant host cellsthat comprise at least one heterologous polynucleotide encoding apolypeptide having DHAD activity and at least one heterologouspolynucleotide encoding a polypeptide affecting Fe—S clusterbiosynthesis. For example, lactic acid bacteria comprising recombinantDHAD and at least one recombinant genetic expression element encodingFe—S cluster forming proteins are the subject of U.S. application Ser.No. 12/569,103, filed Sep. 29, 2009, which is incorporated by referenceherein. The present recombinant host cells comprising at least oneheterologous polynucleotide encoding a polypeptide having DHAD activityand at least one heterologous polynucleotide encoding a polypeptideaffecting Fe—S cluster biosynthesis do not include those lactic acidbacteria described in U.S. application Ser. No. 12/569,103, filed Sep.29, 2009, which is incorporated by reference herein.

The polypeptide affecting Fe—S cluster biosynthesis can be selected fromthe group consisting of the Fe uptake and utilization or Fe—S clusterbiosynthesis pathway genes in Tables 7, 8 and 9. In one embodiment, thepolypeptide affecting Fe—S cluster biosynthesis is encoded by ARN1,ARN2, ATX1, CCC2, COT1, ENB1, FET3, FET5, FIT1, FIT2, FIT3, FRE1, FRE2,FRE3, FRE4, FRE5, FREE, FTH1, FTR1, HMX1, SIT1, SMF3, TIS11, VHT1, AFT1,AFT2, AIM1, ARH1, ATM1, BUD32, CAD1, CCC1, CFD1, CIA1, CMK1, CTH1, CTI6,CYC8, DAP1, DRE2, ERV1, ESA1, FET4, FRA1, FRA2, GEF1, GGC1, GRX1, GRX2,GRX4, GRX5, HDA1, IBA57, ISA1, ISA2, ISU1, ISU2, JAC1, MGE1, MRS3, MRS4,MSN5, NAR1, NFS1, NFU1, NHP6a, NHP6b, PSE1, SMF1, SNF1, SNF2, SNF3,SNF4, SSQ1, TIM12, TUP1, NP_011911.1, VPS41, YAP5, YFH1, YRA1, ZPR1,iscA^(nif), nifU, nifS, cysE1, cysE2, iscS, iscU, iscA, hscB, hscA, Fdx,sufS, sufE, cysE3, sufS2, iscA2, Nfu, nfuA, nfuV, nfu, sufA, sufB, sufC,sufD, sufE1, sufS2, or sufE2. In one embodiment, the polypeptideaffecting Fe—S cluster biosynthesis is AFT1, AFT2, PSE1, FRA2, GRX3, orMSN5. In one embodiment, the polypeptide affecting Fe—S clusterbiosynthesis is selected from the group consisting of AFT1, AFT2, PSE1,FRA2, GRX3, MSN5, and combinations thereof. In one embodiment, thepolypeptide affecting Fe—S cluster biosynthesis is selected from thegroup consisting of AFT1, AFT2, PSE1, FRA2, MSN5, and combinationsthereof. In another embodiment, the polypeptide affecting Fe—S clusterbiosynthesis is selected from the group consisting of AFT1, AFT2, PSE1,FRA2, GRX3, MSN5, and combinations thereof, and the polypeptideaffecting Fe—S cluster biosynthesis is encoded by a polynucleotidecomprising a plasmid. In some embodiments, DHAD is co-expressed withAFT1, AFT2, PSE1, and combinations thereof. The polypeptide affectingFe—S cluster biosynthesis may be a constitutive mutant, such as, but notlimited to, AFT1 L99A, AFT1 L102A, AFT1 C291F, AFT1 C293F, andcombinations thereof. The deletion, mutation, and/or substitution in theendogenous gene encoding a polypeptide affecting Fe—S clusterbiosynthesis can be selected from the group consisting of FRA2, GRX3,MSN5, and combinations thereof.

The present invention also provides a method for increasing the activityof an Fe—S cluster requiring protein in a recombinant host cellcomprising providing a recombinant host cell comprising an Fe—S clusterrequiring protein, changing the expression or activity of a polypeptideaffecting Fe—S cluster biosynthesis in the host cell, and growing therecombinant host cell with the changed expression or activity underconditions whereby the activity of the Fe—S cluster requiring protein isincreased. Such a method can be used to increase the activity of anendogenous Fe—S cluster requiring protein, or a heterologous Fe—Scluster requiring protein. Such a method can be used to increase thespecific activity of a DHAD described herein, or identified by themethods described herein. The increase in the activity of the Fe—Scluster requiring protein can be in an amount selected from greater thanabout 10%; greater than about 15%; greater than about 20%; greater thanabout 25%; greater than about 30%; greater than about 35%; greater thanabout 40%; greater than about 45%; greater than about 50%; greater thanabout 55%; greater than about 60%; greater than about 65%; greater thanabout 70%; greater than about 75%; greater than about 80%; greater thanabout 85%; greater than about 90%; and greater than about 95%. Theincrease in activity may be greater than about 3 fold, greater thanabout 5 fold, greater than about 8 fold, or greater than about 10 fold.In embodiments, the activity of the Fe—S cluster requiring protein canbe in an amount that is at least about 60% of theoretical, at leastabout 70% of theoretical, at least about 80% theoretical, or at leastabout 90% theoretical.

The present invention can also be used to increase the flux in the Fe—Scluster biosynthesis pathway in a host cell and to identify polypeptidesthat increase the flux in an Fe—S cluster biosynthesis pathway in a hostcell. In one embodiment a method is provided for increasing the flux inan Fe—S cluster biosynthesis pathway in a host cell comprising providinga recombinant host cell comprising an Fe—S cluster requiring protein andeither at least one polypeptide affecting Fe—S cluster biosynthesis, atleast one deletion, mutation, and/or substitution in an endogenous geneencoding a polypeptide affecting Fe—S cluster biosynthesis, or acombination of both, and growing the recombinant host cell underconditions whereby the flux in the Fe—S cluster biosynthesis pathway inthe host cell is increased. In another embodiment, a method is providedfor identifying polypeptides that increase the flux in an Fe—S clusterbiosynthesis pathway in a host cell comprising: (a) changing theexpression or activity of a polypeptide affecting Fe—S clusterbiosynthesis; (b) measuring the activity of a Fe—S cluster requiringprotein; and (c) comparing the activity of the Fe—S cluster requiringprotein measured in the presence of the change in expression or activitypolypeptide of step (a) to the activity of the Fe—S cluster requiringprotein measured in the absence of the change in expression or activitypolypeptide of step (a), wherein an increase in the activity of theheterologous Fe—S cluster requiring protein indicates an increase in theflux in said Fe—S cluster biosynthesis pathway. In such methods, theFe—S cluster requiring protein may be endogenous or heterologous to thehost cell.

The expression or activity of the polypeptide affecting Fe—S clusterbiosynthesis can be changed by methods well known in the art, including,but not limited to, deleting, mutating, substituting, expressing,up-regulating, down-regulating, altering the cellular location, alteringthe state of the protein, and/or adding a cofactor, and combinationsthereof. Altering the state of the protein can include, but are notlimited to, such alterations as phosphorylation or ubiquitination. Anynumber of methods described herein or known in the art can be used tomeasure the activity of the Fe—S cluster requiring protein, dependingupon the Fe—S cluster requiring protein chosen. For example, if DHAD isthe Fe—S cluster requiring protein, the assay described in the Example 7can be used to measure the activity of the DHAD to determine if there isan increase in the flux in the Fe—S cluster biosynthesis pathway of thehost cell.

Isobutanol and Other Products

Expression of a DHAD in a recombinant host cell, as described herein,provides the transformed, recombinant host cell with dihydroxy-aciddehydratase activity for conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate or 2,3-dihydroxymethylvalerate toα-ketomethylvalerate. A product that has α-ketoisovalerate orα-ketomethylvalerate as a pathway intermediate may be produced withgreater effectiveness in a host cell disclosed herein having thedescribed heterologous DHAD. A list of such products includes, but isnot limited to, valine, isoleucine, leucine, pantothenic acid,2-methyl-1-butanol, 3-methyl-1-butanol, and isobutanol.

For example, biosynthesis of valine in yeast includes steps ofacetolactate conversion to 2,3-dihydroxy-isovalerate by acetohydroxyacidreductoisomerase (ILV5), conversion of 2,3-dihydroxy-isovalerate toα-ketoisovalerate (also called 2-ketoisovalerate) by dihydroxy-aciddehydratase, and conversion of α-ketoisovalerate to valine bybranched-chain amino acid transaminase (BAT2) and branched-chain aminoacid aminotransferase (BAT1). Biosynthesis of leucine includes the samesteps to α-ketoisovalerate, followed by conversion of α-ketoisovalerateto alpha-isopropylmalate by alpha-isopropylmalate synthase (LEU9, LEU4),conversion of alpha-isopropylmalate to beta-isopropylmalate byisopropylmalate isomerase (LEU1), conversion of beta-isopropylmalate toalpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finallyconversion of alpha-ketoisocaproate to leucine by branched-chain aminoacid transaminase (BAT2) and branched-chain amino acid aminotransferase(BAT1). The bacterial pathway is similar, involving differently namedproteins and genes. Increased conversion of 2,3-dihydroxy-isovalerate toα-ketoisovalerate will increase flow in these pathways, particularly ifone or more additional enzymes of a pathway is overexpressed. Thus, itis desired for production of valine or leucine to use a strain disclosedherein.

Biosynthesis of pantothenic acid includes a step performed by DHAD, aswell as steps performed by ketopantoate hydroxymethyltransferase andpantothenate synthase. Engineering of expression of these enzymes forenhanced production of pantothenic acid biosynthesis in microorganismsis described in U.S. Pat. No. 6,177,264.

The α-ketoisovalerate product of DHAD is an intermediate in isobutanolbiosynthetic pathways disclosed in U.S. Patent Appl. Pub. No.20070092957 A1, which is incorporated by reference herein. A diagram ofdisclosed isobutanol biosynthetic pathways is provided in FIG. 5.Production of isobutanol in a strain disclosed herein may benefit fromincreased DHAD activity. As disclosed herein, increased DHAD activity isprovided by expression of a DHAD in a host cell, for example, byover-expressing the DHAD, by modulating the expression or activity of apolypeptide having Fe—S cluster regulatory activity, or a combination ofboth expression of a DHAD and modulation of the expression or activityof a polypeptide having Fe—S cluster regulatory activity. As describedin U.S. Patent Appl. Pub. No. 20070092957 A1, which is incorporated byreference herein, steps in an example isobutanol biosynthetic pathwayinclude conversion of:

-   -   pyruvate to acetolactate (see FIG. 5, pathway step a therein),        as catalyzed for example by acetolactate synthase,    -   acetolactate to 2,3-dihydroxyisovalerate (see FIG. 5, pathway        step b therein) as catalyzed for example by acetohydroxy acid        isomeroreductase;    -   2,3-dihydroxyisovalerate to α-ketoisovalerate (see FIG. 5,        pathway step c therein) as catalyzed for example by acetohydroxy        acid dehydratase, also called dihydroxy-acid dehydratase (DHAD);    -   α-ketoisovalerate to isobutyraldehyde (see FIG. 5, pathway step        d therein) as catalyzed for example by branched-chain α-keto        acid decarboxylase; and    -   isobutyraldehyde to isobutanol (see FIG. 5, pathway step e        therein) as catalyzed for example by branched-chain alcohol        dehydrogenase.

The substrate to product conversions, and enzymes involved in thesereactions, for steps f, g, h, I, j, and k of alternative pathways aredescribed in U.S. Patent Appl. Pub. No. 20070092957 A1, which isincorporated by reference herein.

Genes that can be used for expression of the pathway step enzymes namedabove other than the DHADs disclosed herein, as well as those foradditional isobutanol pathways, are described in U.S. Patent Appl. Pub.No. 20070092957 A1, which is incorporated by reference herein.Additional genes that may be used can be identified by one skilled inthe art through bioinformatics or using methods well-known in the art,such as the various methods described in U.S. application Ser. No.12/569,636, filed Sep. 29, 2009, which is incorporated by referenceherein, to isolate homologs. Suitable ketol-acid reductoisomerase (KARI)enzymes are described in U.S. Patent Appl. Pub. Nos. 20080261230 A1,20090163376, 20100197519, and U.S. application Ser. No. 12/893,077, allincorporated by reference herein. Examples of KARIs disclosed thereinare those from Vibrio cholerae, Pseudomonas aeruginosa PAO1, andPseudomonas fluorescens PF5. U.S. Patent Appl. Publ. No. 2009/0269823and U.S. Prov. Patent Appl. No. 61/290,636, incorporated by referenceherein, describe suitable alcohol dehydrogenases.

Additionally described in U.S. Patent Appl. Pub. No. 20070092957 A1,which is incorporated by reference herein, are construction of chimericgenes and genetic engineering of bacteria and yeast for isobutanolproduction using the disclosed biosynthetic pathways.

Additional Modifications

Examples of additional modifications that may be useful in cellsprovided herein include modifications to reduce glycerol-3-phosphatedehydrogenase activity and/or disruption in at least one gene encoding apolypeptide having pyruvate decarboxylase activity or a disruption in atleast one gene encoding a regulatory element controlling pyruvatedecarboxylase gene expression as described in U.S. Patent Appl. Pub. No.20090305363 (incorporated herein by reference), modifications to a hostcell that provide for increased carbon flux through an Entner-DoudoroffPathway or reducing equivalents balance as described in U.S. PatentAppl. Pub. No. 20100120105 (incorporated herein by reference). Othermodifications include integration of at least one polynucleotideencoding a polypeptide that catalyzes a step in a pyruvate-utilizingbiosynthetic pathway described in U.S. Prov. Appl. No. 61/380,563(incorporated herein by reference). Additional modifications that may besuitable are described in U.S. application. Ser. No. 12/893,089.Additionally, host cells comprising a heterologous polynucleotideencoding a polypeptide with phosphoketolase activity and host cellscomprising a heterologous polynucleotide encoding a polypeptide withphosphotransacetylase activity are described in U.S. Provisional PatentApplication No. 61/356,379.

Growth for Production

Recombinant host cells disclosed herein are grown in fermentation mediawhich contains suitable carbon substrates. Suitable carbon substratesmay include, but are not limited to, monosaccharides such as glucose,fructose, oligosaccharides such as lactose maltose, galactose, orsucrose, polysaccharides such as starch or cellulose or mixtures thereofand unpurified mixtures from renewable feedstocks such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt. Othercarbon substrates may include ethanol, lactate, succinate, or glycerol.

Additionally the carbon substrate may also be one-carbon substrates suchas carbon dioxide, or methanol for which metabolic conversion into keybiochemical intermediates has been demonstrated. Two-carbon substratessuch as ethanol may also suitable. In addition to one and two carbonsubstrates, methylotrophic organisms are also known to utilize a numberof other carbon containing compounds such as methylamine, glucosamineand a variety of amino acids for metabolic activity. For example,methylotrophic yeasts are known to utilize the carbon from methylamineto form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153:485-489 (1990)). Hence it is contemplated that the sourceof carbon utilized in the present invention may encompass a wide varietyof carbon containing substrates and will only be limited by the choiceof organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,in some embodiments, the carbon substrates are glucose, fructose, andsucrose, or mixtures of these with C5 sugars such as xylose and/orarabinose for yeasts cells modified to use C5 sugars. Sucrose may bederived from renewable sugar sources such as sugar cane, sugar beets,cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose maybe derived from renewable grain sources through saccharification ofstarch based feedstocks including grains such as corn, wheat, rye,barley, oats, and mixtures thereof. In addition, fermentable sugars maybe derived from renewable cellulosic or lignocellulosic biomass throughprocesses of pretreatment and saccharification, as described, forexample, in co-owned and co-pending U.S. Patent Appl. Pub. No.20070031918 A1, which is herein incorporated by reference. Biomassrefers to any cellulosic or lignocellulosic material and includesmaterials comprising cellulose, and optionally further comprisinghemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.Biomass may also comprise additional components, such as protein and/orlipid. Biomass may be derived from a single source, or biomass cancomprise a mixture derived from more than one source; for example,biomass may comprise a mixture of corn cobs and corn stover, or amixture of grass and leaves. Biomass includes, but is not limited to,bioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. Examples of biomass include, but are not limited to,corn grain, corn cobs, crop residues such as corn husks, corn stover,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animalmanure, and mixtures thereof.

In addition to an appropriate carbon source, growth media must containsuitable minerals, salts, cofactors, buffers and other components, knownto those skilled in the art, suitable for the growth of the cultures andpromotion of an enzymatic pathway comprising a Fe—S cluster requiringprotein such as, for example, DHAD.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C.to about 40° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth, Yeast Medium (YM)broth, or broth that includes yeast nitrogen base, ammonium sulfate, anddextrose (as the carbon/energy source) or YPD Medium, a blend ofpeptone, yeast extract, and dextrose in optimal proportions for growingmost Saccharomyces cerevisiae strains. Other defined or synthetic growthmedia may also be used, and the appropriate medium for growth of theparticular microorganism will be known by one skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate, may also be incorporated into the growthmedium.

Suitable pH ranges for the growth are between about pH 5.0 to about pH9.0. In one embodiment, about pH 6.0 to about pH 8.0 is used for theinitial condition. Suitable pH ranges for the fermentation of yeast aretypically between about pH 3.0 to about pH 9.0. In one embodiment, aboutpH 5.0 to about pH 8.0 is used for the initial condition. Suitable pHranges for the fermentation of other microorganisms are between about pH3.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 isused for the initial condition.

Growth may be performed under aerobic or anaerobic conditions. In oneembodiment, anaerobic or microaerobic conditions are used for growth.

Industrial Batch and Continuous Fermentations

Isobutanol, or other products, may be produced using a batch method offermentation. A classical batch fermentation is a closed system wherethe composition of the medium is set at the beginning of thefermentation and not subject to artificial alterations during thefermentation. A variation on the standard batch system is the fed-batchsystem. Fed-batch fermentation processes are also suitable in thepresent invention and comprise a typical batch system with the exceptionthat the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression isapt to inhibit the metabolism of the cells and where it is desirable tohave limited amounts of substrate in the media. Batch and fed-batchfermentations are common and well known in the art and examples may befound in Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol.,36:227, (1992), herein incorporated by reference.

Isobutanol, or other products, may also be produced using continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.Methods of modulating nutrients and growth factors for continuousfermentation processes as well as techniques for maximizing the rate ofproduct formation are well known in the art of industrial microbiologyand a variety of methods are detailed by Brock, supra.

It is contemplated that the production of isobutanol, or other products,may be practiced using batch, fed-batch or continuous processes and thatany known mode of fermentation would be suitable. Additionally, it iscontemplated that cells may be immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isobutanolproduction.

Methods for Isobutanol Isolation from the Fermentation Medium

Bioproduced isobutanol may be isolated from the fermentation mediumusing methods known in the art for ABE fermentations (see, e.g., Durre,Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process.Biochem. 27:61-75 (1992), and references therein). For example, solidsmay be removed from the fermentation medium by centrifugation,filtration, decantation, or the like. Then, the isobutanol may beisolated from the fermentation medium using methods such asdistillation, azeotropic distillation, liquid-liquid extraction,adsorption, gas stripping, membrane evaporation, or pervaporation.

Because isobutanol forms a low boiling point, azeotropic mixture withwater, distillation can be used to separate the mixture up to itsazeotropic composition. Distillation may be used in combination withanother separation method to obtain separation around the azeotrope.Methods that may be used in combination with distillation to isolate andpurify butanol include, but are not limited to, decantation,liquid-liquid extraction, adsorption, and membrane-based techniques.Additionally, butanol may be isolated using azeotropic distillationusing an entrainer (see, e.g., Doherty and Malone, Conceptual Design ofDistillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so thatdistillation may be used in combination with decantation to isolate andpurify the isobutanol. In this method, the isobutanol containingfermentation broth is distilled to near the azeotropic composition.Then, the azeotropic mixture is condensed, and the isobutanol isseparated from the fermentation medium by decantation. The decantedaqueous phase may be returned to the first distillation column asreflux. The isobutanol-rich decanted organic phase may be furtherpurified by distillation in a second distillation column.

The isobutanol may also be isolated from the fermentation medium usingliquid-liquid extraction in combination with distillation. In thismethod, the isobutanol is extracted from the fermentation broth usingliquid-liquid extraction with a suitable solvent. Theisobutanol-containing organic phase is then distilled to separate thebutanol from the solvent.

Distillation in combination with adsorption may also be used to isolateisobutanol from the fermentation medium. In this method, thefermentation broth containing the isobutanol is distilled to near theazeotropic composition and then the remaining water is removed by use ofan adsorbent, such as molecular sieves (Aden et al. LignocellulosicBiomass to Ethanol Process Design and Economics Utilizing Co-CurrentDilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover,Report NREL/TP-510-32438, National Renewable Energy Laboratory, June2002).

Additionally, distillation in combination with pervaporation may be usedto isolate and purify the isobutanol from the fermentation medium. Inthis method, the fermentation broth containing the isobutanol isdistilled to near the azeotropic composition, and then the remainingwater is removed by pervaporation through a hydrophilic membrane (Guo etal., J. Membr. Sci. 245, 199-210 (2004)).

Embodiments of the Inventions

Embodiment 1 (E1). A recombinant host cell comprising at least oneheterologous polynucleotide encoding a polypeptide having dihydroxy-aciddehydratase activity wherein said at least one heterologouspolynucleotide comprises a high copy number plasmid or a plasmid with acopy number that can be regulated.

E2. A recombinant host cell comprising at least one heterologouspolynucleotide encoding a polypeptide having dihydroxy-acid dehydrataseactivity wherein said at least one heterologous polynucleotide isintegrated at least once in the recombinant host cell DNA.

E3. A recombinant host cell comprising at least one heterologouspolynucleotide encoding a polypeptide having dihydroxy-acid dehydrataseactivity, wherein said host cell comprises at least one deletion,mutation, and/or substitution in an endogenous gene encoding apolypeptide affecting Fe—S cluster biosynthesis.

E4. A recombinant host cell comprising at least one heterologouspolynucleotide encoding a polypeptide having dihydroxy-acid dehydrataseactivity and at least one heterologous polynucleotide encoding apolypeptide affecting Fe—S cluster biosynthesis.

E5. The recombinant host cell of any one of embodiments E3-E4, whereinsaid heterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis is selected from the group consisting of the genesin Tables 8 and 9.

E6. The recombinant host cell of any one of embodiments E3-E4, whereinsaid heterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis is selected from the group consisting of the genesin Table 7.

E7. The recombinant host cell of embodiment E5 or E6, wherein saidheterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis is selected from the group consisting of AFT1,AFT2, PSE1, FRA2, GRX3, MSN5, and combinations thereof.

E8. The recombinant host cell of embodiment E7, wherein said polypeptideis encoded by a polynucleotide that is constitutive mutant.

E9. The recombinant host cell of embodiment E8, wherein saidconstitutive mutant is selected from the group consisting of AFT1 L99A,AFT1 L102A, AFT1 C291F, AFT1 C293F, and combinations thereof.

E10. The recombinant host cell of embodiment E7, wherein saidpolypeptide affecting Fe—S cluster biosynthesis is encoded by apolynucleotide comprising a high copy number plasmid or a plasmid with acopy number that can be regulated.

E11. The recombinant host cell of embodiment E7, wherein saidpolypeptide affecting Fe—S cluster biosynthesis is encoded by apolynucleotide integrated at least once in the recombinant host cellDNA.

E12. The recombinant host cell of embodiment E3, wherein the at leastone deletion, mutation, and/or substitution in an endogenous geneencoding a polypeptide affecting Fe—S cluster biosynthesis is selectedfrom the group consisting of FRA2, GRX3, MSN5, and combinations thereof.

E13. The recombinant host cell of embodiment E4, wherein the at leastone heterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis is selected from the group consisting of AFT1,AFT2, PSE1, and combinations thereof.

E14. The recombinant host cell of any one of embodiments E3-E13, whereinsaid at least one heterologous polynucleotide encoding a polypeptidehaving dihydroxy-acid dehydratase activity is expressed in multiplecopies.

E15. The recombinant host cell of embodiment E14, wherein said at leastone heterologous polynucleotide comprises a high copy number plasmid ora plasmid with a copy number that can be regulated.

E16. The recombinant host cell of embodiment E14, wherein said at leastone heterologous polynucleotide is integrated at least once in therecombinant host cell DNA.

E17. The recombinant host cell of any one of embodiments E3-E16, whereinsaid Fe—S cluster biosynthesis is increased compared to a recombinanthost cell having endogenous Fe—S cluster biosynthesis.

E18. The recombinant host cell of any one of embodiments E1-E17, whereinsaid host cell is a yeast host cell.

E19. The recombinant host cell of embodiment E18, wherein said yeasthost cell is selected from the group consisting of Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia,Issatchenkia and Pichia.

E20. The recombinant host cell of any one of embodiments E1-E19, whereinsaid heterologous polypeptide having dihydroxy-acid dehydratase activityis expressed in the cytosol of the host cell.

E21. The recombinant host cell of any one of embodiments E1-E20, whereinsaid heterologous polypeptide having dihydroxy-acid dehydratase activityhas an amino acid sequence that matches the Profile HMM of Table 12 withan E value of <10⁻⁵ wherein the polypeptide further comprises all threeconserved cysteines, corresponding to positions 56, 129, and 201 in theamino acids sequences of the Streptococcus mutans DHAD enzymecorresponding to SEQ ID NO:168.

E22. The recombinant host cell of any one of embodiments E1-E21, whereinsaid heterologous polypeptide having dihydroxy-acid dehydratase activityhas an amino acid sequence with at least about 90% identity to SEQ IDNO: 168 or SEQ ID NO: 232.

E23. The recombinant host cell of any one of embodiments E1-E22, whereinsaid polypeptide having dihydroxy-acid dehydratase activity has aspecific activity selected from the group consisting of:

-   -   a. greater than about 5-fold with respect to the control host        cell comprising at least one heterologous polynucleotide        encoding a polypeptide having dihydroxy-acid dehydratase        activity;    -   b. greater than about 8-fold with respect to the control host        cell comprising at least one heterologous polynucleotide        encoding a polypeptide having dihydroxy-acid dehydratase        activity; and    -   c. greater than about 10-fold with respect to the control host        cell comprising at least one heterologous polynucleotide        encoding a polypeptide having dihydroxy-acid dehydratase        activity.

E24. The recombinant host cell of any one of embodiments E1-E22, whereinsaid polypeptide having dihydroxy-acid dehydratase activity has aspecific activity selected from the group consisting of:

-   -   a. greater than about 0.25 U/mg;    -   b. greater than about 0.3 U/mg;    -   c. greater than about 0.5 U/mg;    -   d. greater than about 1.0 U/mg;    -   e. greater than about 1.5 U/mg;    -   f. greater than about 2.0 U/mg;    -   g. greater than about 3.0 U/mg;    -   h. greater than about 4.0 U/mg;    -   i. greater than about 5.0 U/mg;    -   j. greater than about 6.0 U/mg;    -   k. greater than about 7.0 U/mg;    -   l. greater than about 8.0 U/mg;    -   m. greater than about 9.0 U/mg;    -   n. greater than about 10.0 U/mg;    -   o. greater than about 20.0 U/mg; and    -   p. greater than about 50.0 U/mg.

E25. The recombinant host cell of any one of embodiments E1-E24, whereinsaid recombinant host cell produces isobutanol.

E26. The recombinant host cell of embodiment E25, wherein saidrecombinant host cell comprises an isobutanol biosynthetic pathway.

E27. A method of making a product comprising:

-   -   a. providing the recombinant host cell of any one of embodiments        E1-E24; and    -   b. contacting the recombinant host cell of (a) with a        fermentable carbon substrate in a fermentation medium under        conditions wherein said product is produced; wherein the product        is selected from the group consisting of branched chain amino        acids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol,        isobutanol, and combinations thereof.

E28. A method of making isobutanol comprising:

-   -   a. providing the recombinant host cell of any one of embodiments        E1-E24;    -   b. contacting the recombinant host cell of (a) with a        fermentable carbon substrate in a fermentation medium under        conditions wherein isobutanol is produced.

E29. A method for the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate comprising:

-   -   a. providing the recombinant host of any one of embodiments        E1-E24;    -   b. growing the recombinant host cell of (a) under conditions        where the 2,3-dihydroxyisovalerate is converted to        α-ketoisovalerate, wherein 2,3-dihydroxyisovalerate is converted        to α-ketoisovalerate.

E30. A method for increasing the specific activity of a heterologouspolypeptide having dihydroxy-acid dehydratase activity in a recombinanthost cell comprising:

-   -   a. providing a recombinant host cell of any one of embodiments        E1-E24; and    -   b. growing the recombinant host cell of (a) under conditions        whereby the heterologous polypeptide having dihydroxy-acid        dehydratase activity is expressed in functional form having a        specific activity greater than the same host cell lacking said        heterologous polypeptide.

E31. A method for increasing the flux in an Fe—S cluster biosynthesispathway in a host cell comprising:

-   -   a. providing a recombinant host cell of any one of embodiments        E3-E24; and    -   b. growing the recombinant host cell of (a) under conditions        whereby the flux in the Fe—S cluster biosynthesis pathway in the        host cell is increased.

E32. A method of increasing the activity of an Fe—S cluster requiringprotein in a recombinant host cell comprising:

-   -   a. providing a recombinant host cell comprising an Fe—S cluster        requiring protein;    -   b. changing the expression or activity of a polypeptide        affecting Fe—S cluster biosynthesis in said host cell; and    -   c. growing the recombinant host cell of (b) under conditions        whereby the activity of the Fe—S cluster requiring protein is        increased.

E33. The method of embodiment E32, wherein said increase in activity isan amount selected from the group consisting of:

-   -   a. greater than about 10%;    -   b. greater than about 20%;    -   c. greater than about 30%;    -   d. greater than about 40%;    -   e. greater than about 50%;    -   f. greater than about 60%;    -   g. greater than about 70%;    -   h. greater than about 80%;    -   i. greater than about 90%; and    -   j. greater than about 95%.

E34. The method of embodiment E32, wherein said increase in activity isan amount selected from the group consisting of:

-   -   a. greater than about 5 fold;    -   b. greater than about 8 fold;    -   c. greater than about 10 fold.

E35. A method for identifying polypeptides that increase the flux in anFe—S cluster biosynthesis pathway in a host cell comprising:

-   -   a. changing the expression or activity of a polypeptide        affecting Fe—S cluster biosynthesis;    -   b. measuring the activity of a heterologous Fe—S cluster        requiring protein; and    -   c. comparing the activity of the heterologous Fe—S cluster        requiring protein measured in the presence of the changed        expression or activity of a polypeptide of step (a) to the        activity of the heterologous Fe—S cluster requiring protein        measured in the absence of the changed expression or activity of        a polypeptide of step (a),    -   wherein an increase in the activity of the heterologous Fe—S        cluster requiring protein indicates an increase in the flux in        said Fe—S cluster biosynthesis pathway.

E36. A method for identifying polypeptides that increase the flux in anFe—S cluster biosynthesis pathway in a host cell comprising:

-   -   a. changing the expression or activity of a polypeptide        affecting Fe—S cluster biosynthesis;    -   b. measuring the activity of a polypeptide having dihydroxy-acid        dehydratase activity; and    -   c. comparing the activity of the polypeptide having        dihydroxy-acid dehydratase activity measured in the presence of        the change in expression or activity of a polypeptide of        step (a) to the activity of the polypeptide having        dihydroxy-acid dehydratase activity measured in the absence of        the change in expression or activity of a polypeptide of step        (a),    -   wherein an increase in the activity of the polypeptide having        dihydroxy-acid dehydratase activity indicates an increase in the        flux in said Fe—S cluster biosynthesis pathway.

E37. The method of any one of embodiments E30-E36, wherein said changingthe expression or activity of a polypeptide affecting Fe—S clusterbiosynthesis comprises deleting, mutating, substituting, expressing,up-regulating, down-regulating, altering the cellular location, alteringthe state of the protein, and/or adding a cofactor.

E38. The method of any one of embodiments E32-E37, wherein the Fe—Scluster requiring protein has dihydroxy-acid dehydratase activity andwherein said Fe—S cluster requiring protein having dihydroxy-aciddehydratase activity has an amino acid sequence that matches the ProfileHMM of Table 12 with an E value of <10⁻⁵ wherein the polypeptide furthercomprises all three conserved cysteines, corresponding to positions 56,129, and 201 in the amino acids sequences of the Streptococcus mutansDHAD enzyme corresponding to SEQ ID NO:168.

E39. The method of any one of embodiments E32-E38, wherein saidpolypeptide affecting Fe—S cluster biosynthesis is selected from thegroup consisting of the genes in Tables 7, 8 and 9.

E40. A recombinant host cell comprising at least one polynucleotideencoding a polypeptide identified by the methods of any one ofembodiments E35-E37.

E41. The recombinant host cell of embodiment E40, wherein said host cellfurther comprises at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity.

E42. The recombinant host cell of embodiment E41, wherein saidheterologous polynucleotide encoding a polypeptide having dihydroxy-aciddehydratase activity is expressed in multiple copies.

E43. The recombinant host cell of embodiment E41, wherein saidheterologous polynucleotide comprises a high copy number plasmid or aplasmid with a copy number that can be regulated.

E44. The recombinant host cell of embodiment E41, wherein saidheterologous polynucleotide is integrated at least once in therecombinant host cell DNA.

E45. The method of embodiment E35 or E36, wherein said host cell is ayeast host cell.

E46. The method of embodiment E45, wherein said yeast host cell isselected from the group consisting of Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia,Issatchenkia, and Pichia.

E47. The method of any one of embodiments E28-E39, wherein said hostcell is a yeast host cell.

E48. The method of embodiment E47, wherein said yeast host cell isselected from the group consisting of Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia,Issatchenkia, and Pichia.

E49. The recombinant host cell of any one of embodiments E40-E44,wherein said recombinant host cell is a yeast host cell.

E50. The recombinant host cell of embodiment E49, wherein said yeasthost cell is selected from the group consisting of Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia,Issatchenkia, and Pichia.

E51. The recombinant host cell of any one of embodiments E40-E44 orE49-E50, wherein said heterologous polypeptide having dihydroxy-aciddehydratase activity is expressed in the cytosol of the host cell.

E52. The recombinant host cell of any one of embodiments E40-E44 orE49-E50, wherein said heterologous polypeptide having dihydroxy-aciddehydratase activity has an amino acid sequence that matches the ProfileHMM of Table 12 with an E value of <10⁻⁵ wherein the polypeptide furthercomprises all three conserved cysteines, corresponding to positions 56,129, and 201 in the amino acids sequences of the Streptococcus mutansDHAD enzyme corresponding to SEQ ID NO:168.

E53. The recombinant host cell of any one of embodiments E40-E44 orE49-E50, wherein said recombinant host cell produces a product selectedfrom the group consisting of branched chain amino acids, pantothenicacid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, andcombinations thereof.

E54. The recombinant host cell of embodiment E53, wherein saidrecombinant host cell produces isobutanol.

E55. The recombinant host cell of embodiment E54, wherein saidrecombinant host cell comprises an isobutanol biosynthetic pathway.

EXAMPLES

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “sec” means second(s), “μl” means microliter(s), “ml”means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm”means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s),“mM” means millimolar, “M” means molar, “mmol” means millimole(s),“μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s),“mg” means milligram(s), “rpm” means revolutions per minute, “w/v” meansweight/volume, “OD” means optical density, and “OD₆₀₀” means opticaldensity measured at a wavelength of 600 nm.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984,and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or by Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass., 1989. All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), LifeTechnologies (Rockville, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

Example 1 Over-expression of DHAD Protein Encoded by the ilvD Gene fromS. mutans using a Plasmid-based System in Yeast Cytosol

Over-expression of a recombinant polynucleotide can be accomplished byincreasing the copy number of a plasmid comprising the recombinantpolynucleotide. To over-express the DHAD protein in yeast, an induciblevector was constructed. The pHR81 vector contains a Ura3 marker as wellas a LEU marker with a defective promoter (see U.S. Patent Appl. Pub.No. 2007/0092957, which is incorporated by reference herein). When theyeast synthetic dropout (SD; also known as complete minimal media;Teknova) growth medium is switched from SD minus uracil to SD minusleucine, the copy number of the pHR81 plasmid increases, resulting inmuch higher level of expression of the recombinant polynucleotide. ThepHR81 vector backbone was derived from pLH472 JEG4y (SEQ ID NO: 921) andwas prepared by digesting the pLH472 JEG4y vector with SpeI and SacII.

For over-expression of a DHAD protein, the DHAD gene ilvD from S. mutans(SEQ ID NO:167) was used (see U.S. Published Patent Appl. No.US2009-0305363A1, which is incorporated by reference herein). This genehas been cloned under the control of the FBA promoter in vector pRS423FBA ilvD Strep-lumio (see U.S. Published Patent Appl. No.US2009-0305363A1, which is incorporated by reference herein). The regioncontaining the FBA promoter, the ilvD gene, and FBA terminator cassettewas amplified with primer set FBAp-F(NheI) and FBAt-R(SacII) (SEQ IDNOs: 915 and 916) and cloned into the pHR81 vector. The resultingexpression vector was designated as pHR81 FBA-IlvD(Sm) (SEQ ID NO: 917;FIG. 1A).

To over express the S. mutans DHAD protein, the expression vector pHR81FBA-IlvD(Sm) was transformed into wild-type yeast strain BY4741.Transformants were selected on agar plates with SD minus uracil. Forover-expression, yeast strains containing the plasmid were initiallygrown at 30° C. in SD liquid medium minus uracil. A fresh overnightculture (5 ml) was then transferred to a 125 ml flask containing 75 mlof SD medium minus leucine. As a control, another 5 ml of freshovernight culture was transferred into a flask containing 75 ml of SDminus uracil. The cultures were incubated overnight before harvesting bycentrifugation. The DHAD activity was measured in crude extracts ofthese samples using the assay described in Example 7.

The DHAD specific activity obtained in the crude extract in the controlsamples grown in SD minus uracil was in the range of 0.2 to 0.3 U mg⁻¹.The average specific activity obtained from strains grown in the SDmedium minus leucine, however, was 1.6 U mg⁻¹, much higher (˜5 to 8-foldhigher) than the activity from the control samples. DHAD requires Fe—Scluster for its function, and it was not previously known if the nativeyeast Fe—S cluster biosynthesis pathway could accommodate anover-expressed Fe—S cluster requiring protein in yeast cytosol. In aprevious screening experiment using a non-inducible, low-copy numbervector, the DHAD from S. mutans could be recombinantly expressed inyeast cytosol with a specific activity in the range of 0.1 to 0.2 U mg⁻¹in the crude extract (see U.S. patent application Ser. No. 12/569,636,filed on Sep. 29, 2009, which is incorporated by reference herein).Thus, in one embodiment, over-expression of a Fe—S cluster requiringprotein, such as DHAD, in yeast using a high-copy number vector providesincreased specific activity, wherein the specific activity is increasedby at least about 5-fold to at least about 8-fold.

Example 2 Over-Expression of DHAD Protein Encoded by the ilvD Gene fromS. mutans Through Chromosomal Integration

An alternate way to increase the expression of a gene in yeast is tointegrate multiple copies of the gene of interest into the host cell'schromosome. To integrate the ilvD gene from S. mutans (SEQ ID NO:167)into a yeast chromosome, integration vectorpZK-Delta(s)-Leu2-FBA-ilvD(Sm)-FBAt (SEQ ID NO: 918; FIG. 1B) wasconstructed. The integration vector backbone was derived frompSuperscript (Stratagene, La Jolla, Calif.). The S. mutans ilvD gene(nucleotides 1306-3018 of the complement strand) was cloned into theintegration vector under the control of the FBA promoter (nucleotides3026-4023 of the complement strand) so that the ilvD gene would beflanked by a yeast delta sequence (nucleotides 118-267 and 5061-5760 ofthe complement strand). S. cerevisiae contains more than 200 yeast deltasequences (Kim J M et al. Genome Res. 1998; 8:464-478). These deltasequences are targets for multiple integrations. The integration vectorwas also engineered to contain the defective LEU2 marker (nucleotides4100-5191 of the complement strand) for selection of transformed strainswith multiple integration events.

For integration, the vector DNA was linearized with AscI and AatIIdigestion to generate delta sequence flanked strands of vector DNAcomprising the ilvD gene, which were then transformed into the yeaststrain BY4741. Transformants were selected on SD agar medium minusleucine. These transformants were then grown on SD liquid medium minusleucine at 30° C., and the cultures were harvested and analyzed for DHADactivity. The specific activity of DHAD obtained in the crude extractranged from 0.7 to 1.2 U mg⁻¹. This specific activity was about 3- to6-fold higher than that found in BY4741 strains transformed with an ilvDgene-containing plasmid without over-expression

Example 3 Improvement of Specific Activity of DHAD in Yeast DeletionStrains

Although the over-expression strains described in Examples 1 and 2 had ahigh level of activity, not all of the DHAD protein expressed wasactive. For example, the over-expressed DHAD protein accounted forapproximately 5 to 10% of the total cell protein, while yielding aspecific activity of from about 0.7 to 1.6 U mg⁻¹. Given that thespecific activity of the purified DHAD enzyme from S. mutans is 100 Umg⁻¹, expression of DHAD at 10% of total cell protein would be expectedto yield a specific activity upwards of 5 to 10 U mg⁻¹. Although notwishing to be bound by one theory, the difference between the expectedand observed specific activity was likely a result of insufficient Fe—Scluster loading. Thus, increasing Fe—S cluster loading by furthermanipulating the over-expression strains could be used to increase thespecific activity of DHAD.

In order to improve the specific activity, yeast strains with deletionsin genes involved in iron metabolism and Fe—S cluster sensing were usedto investigate their effects on DHAD specific activity. These strains(BY4741 background) were purchased from Open Biosystem (Huntsville,Ala.) and are listed in Table 10. As described in Example 1, the highcopy number plasmid pHR81 FBA-IlvD(Sm) was transformed into thesestrains, and DHAD over-expression was induced by changing the growthmedium to SD minus leucine. Crude extracts from cultures were preparedand assayed for DHAD activity. Results are shown in Table 10.

TABLE 10 Effects of deletions of genes involved in Fe metabolism.Specific Genes Function Activity (U/mg) WT 1.69 ± 0.02 Δisu1 scaffoldprotein for Fe—S cluster assembling 1.31 ± 0.56 Δfra2 repressorcomponent for Aft1p 3.41 ± 0.24 Δsin4 regulatory protein 1.65 ± 0.20Δmtm1 protein involved in metal metabolism 0.54 ± 0.12 Δfra1 regulatoryprotein 0.97 ± 0.05 Δgrx3 glutaredoxins 5.45 ± 0.14 Δaft1 global Feregulator 0.23 ± 0.05 Δaft2 paralogue to Aft1p 1.11 ± 0.38 Δmsn5 nuclearprotein exporter 1.59 ± 0.10 Δfet3 ferrous iron uptake; multi-copperoxidase 0.54 ± 0.09 Δftr1 ferrous iron uptake; permease 0.76 ± 0.03Δccc2 copper transporter (for Fet3p) 1.23 ± 0.17 Δgef1 coppertransporter/loading for Fet3p 1.70 ± 0.10 Δfet4 Low-affinity Fe(II)transporter 1.07 ± 0.02 Δsmf1 Low-affinity Fe(II) transporter 1.78 ±0.12 Δmrs3 mitochondrial iron transporter 1.51 ± 0.13 Δmrs4mitochondrial iron transporter 0.85 ± 0.16 Δcth2 targeted mRNA bindingand degradation 1.28 ± 0.40 Δcth1 targeted mRNA binding and degradation1.44 ± 0.30

Surprisingly, DHAD specific activity in the crude extract in strainswith a deletion in either the FRA2 or the GRX3 gene increased by 2- to3-fold, which was unexpected as many of the deletions tested did notincrease DHAD specific activity. It has been shown that cytosolic ironsulfur assembly (CIA) machinery in yeast is responsible for assembly ofFe—S clusters for cytosolic proteins such as isopropylmalate isomerase(Leu1). Previous results indicate that this CIA machinery is independentfrom the iron sensing system involving Aft1 and a Grx3/Grx4-Fra2heterodimer as the repressor (Rutherford et al, J Biol Chem.280:10135-10140 (2005)).

Another unexpected finding is the effect of a Grx3 deletion on DHADactivity. It has been shown that Grx3 and Grx4 are equivalent infunction. While double mutations in both GRX3 and GRX4 genes resulted indrastic activation of the Fe regulon, mutation in Grx4 alone confersminimal phenotype (Pujol-Carrion, et al, J Cell Sci. 119:4554-4564(2006); Ojeda, et al, J Biol Chem. 281:17661-17669 (2006).). As shown inTable 10 above, GRX3 deletion alone leads to significant improvement inDHAD specific activity.

Thus, these results demonstrate that modulating genes involved in ironmetabolism can increase the activity of an Fe—S cluster requiringprotein such as DHAD when expressed in yeast cytosol. As outlined inFIG. 10, the effect of deletions of the FRA2 and GRX3 genes on DHADspecific activity could result from, e.g., activation of transcriptionof one or more of the genes in the iron regulon via the global regulatorAft1p. Although not wishing to be bound by any one theory, activation ofsuch genes could lead to an increase in iron uptake and an increase incytoplasmic Fe—S cluster biosynthesis, leading to higher Fe—S clusterloading of the protein (FIG. 10). Demonstration of increased Fe—Scluster loading is described in Example 11.

Example 4 Effect of Expression of Aft1p and its Mutants on DHAD SpecificActivity

As described in Example 3 and outlined in FIG. 10, Fra2, Grx3, and Grx4are repressors that regulate the function of Aft1p (Kumánovics, et al.,J. Biol. Chem. 283:10276-10286 (2008)). Aft1p is a global regulator ofiron. Activation of genes involved in iron uptake and metabolismrequires the nuclear localization of Aft1p. Expression of Aft1constitutive mutants or an increase in the expression of wild-typeAft1p, could lead to the activation of the Fe regulon in a wild-typestrain or in an AFT1 deletion strain (Yamaguchi-Iwai, et al, EMBO J.14:1231-1239 (1995); Yamaguchi-Iwai, et al, J. Biol. Chem.277:18914-18918 (2002); Kaplan, et al, Chem. Rev. 109:4536-4552(2009)).Based on the novel findings described in Example 3, it is possible thatexpression of Aft1p protein and its constitutive mutants may improve theactive fraction of the DHAD enzyme which requires Fe—S clusters for itsactivity.

To examine this possibility, the wild-type AFT1 gene and itsconstitutive mutants were cloned using a centromere vector pRS411 (ATCC®Number: 87538; SEQ ID NO: 919). This vector has an ampicillin selectionmarker for growth in E. coli and a methionine nutritional marker forselection in yeast. The wild-type AFT1 gene, including its own promoterand terminator, can be cloned between the KpnI and SacI sites, resultingin the construct pRS411-Aft1+flanking (SEQ ID NO: 920; FIG. 2). Asimilar strategy can be used to clone genes that encode Aft1constitutive mutants. The Aft1 constitutive mutants with substitutionsat amino acids L99 to A and C291 to F (with respect to SEQ ID NO: 703)were first examined. The pRS411 constructs with genes encoding thewild-type AFT1 gene or constitutive mutants were transformed, along withthe expression vector pHR81 FBA IlvD(Sm), into the wild-type yeaststrain BY4741 or a yeast strain with a deletion in AFT1, GRX3, or FRA2.Transformants were selected on agar plates with SD medium minusmethionine and uracil. Transformed strains were grown in SD medium minusmethionine and leucine to over-express the DHAD protein in the presenceof these genes or mutants. The DHAD activity in the crude extract ofthese cultures were measured.

Results of expression of wild-type Aft1p, Aft1p(C291F), and Aft1p(L99A)are shown in Table 11. A moderate increase in DHAD specific activity wasobserved with Aft1p (C291F) as compared to wild-type Aft1p. A muchhigher increase in DHAD activity was observed with Aft1p(L99A). Thespecific activity of DHAD in yeast expressing Aft1p(L99A) was similar tothe specific activity obtained in the GRX3 deletion strain (see Table10).

TABLE 11 Effects of expression of Aft1p and its mutants on the activityof DHAD from S. mutans in Δaft1 strain. Plasmids Specific Activity(U/mg) pHR81-FBA-ilvD(Sm) + pRS411-Aft1 2.60 ± 0.52 pHR81-FBA-ilvD(Sm) +pRS411-Aft1(C291L) 3.79 ± 0.23 pHR81-FBA-ilvD(Sm) + pRS411-Aft1(L99A)5.41 ± 0.41

Example 5 Increase in Cytosolic DHAD Specific Activity in a CCC1Deletion Strain

The exact mechanism of increasing Fe—S cluster biosynthesis capabilityfor cytosolic DHAD protein is unknown. Based on the findings with FRA2and GRX3 deletion strains (Example 3) and with expression of Aft1pmutants (Example 4), increasing the availability of the Fe content inthe cytosol may also improve the DHAD specific activity. CCC1 deletionhas been shown to increase the Fe content of the cytosol (Li L, et al, JBiol Chem. 276:29515-29519 (2001)). To test this hypothesis, the CCC1deletion strain of BY4741 was transformed with plasmid pHR81FBA-IlvD(Sm) as described in Example 1. The crude extracts of cells withthe plasmid were assayed for DHAD activity. Table 13 shows the resultsof the experiment. When the CCC1 deletion strain with the DHAD plasmidwas grown in SD medium lacking uracil, an increase in DHAD specificactivity was found as compared to the wild-type cells with the sameplasmid. When extra Fe was added, a further increase in DHAD wasobserved in the CCC1 deletion strain. Addition of Fe showed no effect onDHAD specific activity in the wild-type cells. To achieve an overexpression of the DHAD protein, strains were grown in SD medium lackingleucine (Example 1). Under these conditions, an increase in DHADspecific activity was detected.

TABLE 13 Expression of DHAD from S. mutans in the BY4741(Δccc1) strain.Strains Growth conditions No extra Fe 100 uM Fe Wild-type -Ura 0.37 ±0.03 0.46 ± 0.04 Δccc1 -Ura 0.83 ± 0.04 1.24 ± 0.03 Wild-type -Leu 1.60± 0.17 1.83 ± 0.31 Δccc1 -Leu 2.53 ± 0.29  2.7 ± 1.07

Example 6 Improvement of Specific Activity of DHAD from L. lactisExpressed in Yeast

Examples 1-5 used the DHAD enzyme from S. mutans to identify novel waysto increase the specific activity of DHAD when expressed in yeast. Inthis example, we investigated the application of these methods toimprove the specific activity of the DHAD enzyme from L. lactis (SEQ IDNO: 958). The IlvD gene from L. lactis (SEQ ID NO: 959) was cloned intothe pHR81 vector under the control of the FBA promoter (FIG. 11). Theresulting construct pHR81 FBA-IlvD(L1)-ADHt (FIG. 11; SEQ ID NO: 960)was transformed into strains with a deletion in either the FRA2 or GRX3gene. To study the effect of the constitutive mutant Aft1p(L99A) on DHADfrom L. lactis, pHR81 FBA-IlvD(L1)-ADHt was co-transformed into yeasthost along with vector pRS411-Aft1(L99A) (see Example 4). Toover-express the IlvD gene, transformants were grown in yeast syntheticdrop-out medium lacking leucine or lacking both leucine and methionine,depending on the strains. Enzymatic assay results are summarized inTable 14. Deletions in FRA2 and GRX3 genes increased the specificactivity of the DHAD from L. lactis when expressed in yeast. Inaddition, expression of the Aft1 constitutive mutant L99A similarlyincreased the specific activity of the DHAD from L. lactis.

TABLE 14 Over-expression of bacterial DHAD from L. lactis in S.cerevisiae. Strains Specific Activity (U/mg) Wild-type 0.23 ± 0.04Δaft1 + Aft1(L99A) 0.95 ± 0.31 Δfra2 0.72 ± 0.04 Δgrx3 0.96 ± 0.05

Example 7 Determining the Specific Activity of DHAD. (Assay Method)

Quantitation of the activity of proteins requiring Fe—S clusters can bedone in an assay format. If the protein is an enzyme, such as DHAD, theactivity is typically expressed in terms of units of activity. A unit ofenzyme activity has been defined by the Enzyme Commission of theInternational Union of Biochemistry as the amount of enzyme that willcatalyze the transformation of 1 micromole of the substrate per minuteunder standard conditions (International Union of Biochemistry, Reportof the Commission on Enzymes, Oxford: Pergamon Press, 1961). Further,the term specific activity is defined as the units of activity in agiven amount of enzyme. Thus, the specific activity is not directlymeasured but is calculated by dividing 1) the activity in units/ml ofthe enzyme sample by 2) the concentration of protein in that sample, sothe specific activity is expressed as units/mg. The specific activity ofa sample of pure, fully active enzyme is a characteristic of thatenzyme. The specific activity of a sample of a mixture of proteins is ameasure of the relative fraction of protein in that sample that iscomposed of the active enzyme of interest. DHAD activity can be measuredspectrophotometrically in an end point assay using the2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D.H. and M. H. Emptage, J. Biol. Chem. 263:3558-64 (1988). In this assay,the 2,4-DNPH reacts with the keto group of the 2-ketoisovaleric acidproduct to form a hydrazone, which is detected by its absorbance at 550nm. The assay buffer contains 50 mM Tris-HCl, 10 mM MgCl₂, pH 8.0 (TM8buffer). Sufficient 2,3-dihydroxyisovaleric acid is added to the assaybuffer so that its final concentration in the assay mix is 10 mM. Ineach assay, an enzyme containing solution and sufficient substratecontaining buffer are mixed so that the final volume is 1 ml. The assaymixture is normally incubated at 37° C. for 30 minutes.

The assay is stopped by adding 250 μl of 10% (W/V) trichloroacetic acid.A few minutes later, 500 μl of a saturated solution of 2,4-DNPH in 1 NHCl is added. The mixture is incubated at room temperature for at least10 min to allow formation of the hydrazone. Next, 1.75 ml of NaOH isadded to solubilize the hydrazone and to precipitate unreacted 2,4-DNPH.A few minutes after the NaOH is added, the assay tubes are placed in asonicator bath for 10 min to degas. The tubes are then centrifuged in adesk top centrifuge at top speed for 2 min to sediment the precipitate.

The absorbance of the supernatant is then read at 550 nm within 1 hour.The absorbance of the sample assays minus the control assays are dividedby 2600 (determined from an α-ketoisovaleric acid standard curve) tofind the units of enzyme activity in the assay. This assay was used inthe Examples described herein in which DHAD specific activity wasdetermined.

Example 8 Purification and Characterization of DHAD from S. mutansExpressed in E. coli

DHAD from S. mutans was purified and characterized as follows. Sixliters of culture of the E. coli Turner strain harboring the pET28aplasmid containing the S. mutans ilvD gene were grown and induced withIPTG. The S. mutans DHAD was purified by breaking the cells with asonicator in TM8 buffer (see Example 7), centrifuging the crude extractto remove cell debris, then loading the supernatant of the crude extracton a Q Sepharose (GE Healthcare) column and eluting the DHAD with anincreasing concentration of NaCl in TM8 buffer. The fractions containingDHAD were pooled, brought to 1 M (NH₄)₂SO₄, and loaded onto aPhenyl-Sepharose column (GE Healthcare) equilibrated with 1 M (NH₄)₂SO₄.The DHAD was eluted with a decreasing concentration of (NH₄)₂SO₄. Thefractions containing DHAD were pooled, concentrated to ≤10 ml, loadedonto a 35×600 cm Superdex-200 column (577 ml bed volume) (GE Healthcare)column, and eluted with TM8 buffer. As judged by SDS gels, the purity ofthe S. mutans DHAD eluted from the Superdex column was estimated to be≥90%.

The UV-visible spectrum of the purified S. mutans DHAD is shown in FIG.3. The number of peaks above 300 nm is typical of proteins with [2Fe-2S]clusters. The S. mutans DHAD was reduced with sodium dithionite, and itsEPR spectra was measured at various temperatures. FIG. 4 shows the EPRspectra measured at temperatures between 20° K and 70° K. The EPRspectrum of the S. mutans DHAD is measureable up to 70° K, whichindicates that it contains a [2Fe-2S] cluster and not a [4Fe-4S] clusterbecause the EPR spectra of proteins containing [4Fe-4S] clusters are notobservable at temperatures much above 10° K.

The exact protein content of the batch of purified S. mutans DHAD withthe highest specific activity using the Bradford protein assay wasdetermined by quantitative amino acid analysis. Combining the activitywith the protein content gave a specific activity of 100 units/mg forthis batch. The iron content of this batch determined by ICP-MS usingmethodology known in the art was 2 molecules of iron per molecule ofDHAD. This is consistent with this batch of S. mutans DHAD containing afull complement of [2Fe-2S] clusters.

Example 9 Separating the Forms of DHAD in Yeast Crude Extract from OtherProteins in the Cell and from Each Other to Measure the Amount of DHADPresent

DHAD protein in yeast cells exists in the forms of dimers with two Fe—Sclusters/dimer, one Fe—S cluster/dimer, and zero Fe—S clusters/dimer. Amethod to measure the concentration of these three forms of DHAD proteinin yeast crude extracts was developed using a Mono Q column and a Source15 PHE PE 4.6/100 column (both columns obtained from GE Healthcare), andis described below.

Frozen yeast cells were thawed, suspended in 50 mM Tris-HCl, 10 mMMgCl₂, pH 8.0 (TM8), then broken by bead beating. The broken cells arecentrifuged to remove the cell debris and generate the yeast crudeextract.

The crude extract was loaded onto a 4 ml Mono Q column attached to anAKTA chromatographic system (GE Healthcare) with the A buffer being TM8and B buffer being TM8 containing 0.5 M NaCl. The column wasequilibrated with A buffer before the sample was loaded. The S. mutansDHAD bound to the Mono Q column under these conditions. After the samplewas loaded onto the column, the column was washed with 10 mL of TM8buffer, then the concentration of NaCl in the eluant was increased to0.22 M NaCl. This was followed by a 30 mL linear gradient from 0.22 M to0.35 M NaCl. During chromatography, the A₂₁₅ of the column eluate wasmonitored, and 1 mL fractions were collected. The fractions were assayedfor DHAD activity. The sum of the activity of the DHAD in the fractionsoff the Mono Q column was close to that in the crude extract. Goodseparations using this column were obtained with as much as 5 mL ofcrude extract representing up to 1 g of yeast cell paste. The DHADcontaining fractions were pooled and made 1.35 M in (NH₄)₂SO₄ inpreparation for chromatography on the PHE column.

The Source 15 PHE PE 4.6/100 column was also attached to an AKTAchromatographic system with the A buffer being TM8 containing 1.5 M(NH₄)₂SO₄ and the B buffer being TM8. Before each run the column wasequilibrated with 90% A. The pooled fractions from the Mono Q columnmade 1.35 M in (NH₄)₂SO₄ were loaded onto the PHE column, and at this(NH₄)₂SO₄ concentration, the DHAD bound to the column. Duringchromatography, the A₂₁₅ of the column eluate was monitored, and 1 mLfractions were collected. The DHAD eluted from the column in three peakswhen the column was developed with a 30 mL decreasing linear gradient of(NH₄)₂SO₄ from 1.35 M to 0 M. The area of each of the DHAD peaks wasdetermined by integration. This elution scheme was found to be ideal forseparating S. mutans DHAD from other yeast proteins that co-eluted withit off the Mono Q column. SDS gels run on fractions where the peakseluted showed that well over 90% of the protein present in these peakswas DHAD when it was expressed at 1% of the soluble protein in yeastcells. The fractions containing each of the three DHAD peaks were pooledseparately. Based on the UV-visible absorbent spectrum and the iron andsulfide contents of the DHAD in these peaks, it was determined that thefirst peak contained DHAD with two [2Fe-2S] clusters/dimers, the secondpeak contained DHAD with one [2Fe-2S] cluster/dimer, and the third peakcontained DHAD with zero [2Fe-2S] clusters/dimers. Thus, in its nativestate, the S. mutans DHAD enzyme appears to exist as a dimer of twomonomeric DHAD proteins.

A standard curve relating the amount of DHAD present in a sample to thesum of the area of the three DHAD peaks off the PHE column was obtainedas follows. Crude extract from yeast cells containing no S. mutans DHADwas spiked with various amounts of purified S. mutans DHAD. Theseextracts were subjected to chromatography on the Mono Q and PHE columnsas described above. The area under each of the three DHAD peaks wasintegrated. The sum of these areas was plotted against the amount ofpure DHAD spiked into the yeast crude extracts. The plot was used toderive the following equation:#μg DHAD in sample of crude extract=0.507×(summed area counts of thethree DHAD peaks)

The DHAD activity in a crude extract of yeast can be readily determinedby the method described in Example 7. The amount of DHAD protein inyeast crude extracts can be determined by the procedure outlined in thisExample. With this data, one can calculate the specific activity of theS. mutans DHAD protein per se in crude extracts according to theprocedure in Example 10.

Example 10 Methods to Determine the Fraction of DHAD in Yeast CrudeExtract Loaded with Fe—S Clusters

When a purified Fe—S cluster requiring protein contains a fullcomplement of clusters, it will have a characteristic specific activity.As previously mentioned, for S. mutans DHAD this specific activity is100 units/mg when it has a full complement of clusters.

A DHAD sample that has on average one Fe—S cluster/per dimer couldcontain some dimers with two clusters, some dimers with one cluster, andsome dimers with no clusters. Alternatively, if cluster addition to adimer is all or none and on average there is one Fe—S cluster/dimer in asample, half of the DHAD dimers would have a full complement of clustersand half would be without clusters. From the results in Example 9, weknow that all or none behavior is not followed by S. mutans DHAD becausea species with one cluster per dimer can be isolated. We have found thatdimers of S. mutans DHAD that have one Fe—S cluster have 1/2 theactivity of dimers with two Fe—S clusters/dimer, i.e., the specificactivity of S. mutans DHAD with 1/2 of a full complement of Fe—Sclusters is 50 units/mg. This means the absence of an Fe—S cluster inone of the monomers of a dimer does not influence the activity of theother monomer should it contain an Fe—S cluster.

With the information obtained with the procedures described in Example 9and the information described so far in this Example, one can determinethe degree of Fe—S cluster loading in a DHAD sample in two differentways.

First, one can compare the ratio of the amounts of the three DHAD peaksto determine the relative amount that has two clusters per dimer, onecluster per dimer, and zero clusters per dimer. This gives the degree ofcluster loading. For example, if the area of peak 1 off the PHE columnwas 25%, peak 2 was 50%, and peak 3 was 25% of the sum of the areas ofpeak 1, peak 2, and peak 3, the percent of the monomers loaded withclusters can be calculated to be 50% according to the followingequation:100*[2*(area of peak 1)+1*(area of peak 2)+0*(area of peak 3)]/[2*(totalpeak area)]=% DHAD monomers with Fe—S clusters.

Second, one can use the specific activity of the DHAD present tocalculate the degree of cluster loading. One determines the specificactivity by dividing the activity determined as described in Example 7with the amount of DHAD protein determined as described in Example 9.The specific activity is then divided by 100 U/mg to determine thefraction of monomers loaded with clusters. This fraction is multipliedby 100 to determine the percent DHAD monomers with Fe—S clusters.

For example if the specific activity is found to be 50 U/mg, thefraction loaded with clusters is 0.5 and the percent DHAD monomers withFe—S clusters is 50%.

To make such a calculation, the specific activity must be based on theconcentration of the DHAD protein in the crude extract (not the totalprotein). Determining the concentration of S. mutans DHAD in thepresence of other proteins can be accomplished using methods describedin Example 9.

Example 11 Specific Activities and Inferred Fraction of the DHAD-LoadedProteins

To determine the fraction of DHAD monomers loaded with Fe—S clusters inseveral yeast strains grown under different conditions, the methodsdescribed above were used. Results are shown in Table 15.

TABLE 15 Specific Activities and Inferred Fraction of the DHAD LoadedProteins. BY DHAD SA in % DHAD is of % Cluster Yeast Growth CrudeExtracts Crude Extract Occupancy of Strain Conditions (U/mg) ProteinDHAD WT -Ura 0.46 2.3 10 ΔFRA2 -Ura 0.8 2.5 14 ΔGRX3 -Ura 0.99 2.4 23 WT-Leu 0.82 11 7 ΔFRA2 -Leu 2.2 11 19 ΔGRX3 -Leu 3.5 9.5 31

These results indicate that under these growth conditions, the level ofFe—S cluster loading in the DHAD in strains lacking FRA2 and GRX3 ishigher than in strains containing functional copies of these genes.Thus, a higher fraction of the DHAD protein is in the active form in thedeletion strains because the content of Fe—S clusters (which arerequired for activity) is higher.

Example 12 Construction of Saccharomyces cerevisiae Strains PNY1505,PNY1541, and PNY1542

The purpose of this Example was to construct Saccharomyces cerevisiaestrains PNY1505, PNY1541, and PNY1542. These strains were derived fromPNY1503 (BP1064). PNY1503 was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry Centre,Netherlands). The construction of PNY1503 (BP1064) is described in U.S.Appl. No. 61/368,436, incorporated by reference herein, and in Example13 below. PNY1505 contains a deletion of the FRA2 gene. PNY1541 andPNY1542 contain an integration of the AFT/gene with the L99A mutation(AFT1-L99A) at the YPRCΔ15 locus.

Deletions/integrations were created by homologous recombination with PCRfragments containing regions of homology upstream and downstream of thetarget gene and the URA3 gene for selection of transformants. The URA3gene was removed by homologous recombination to create a scarlessdeletion/integration.

The scarless deletion/integration procedure was adapted from Akada etal., Yeast, 23(5):399-405 (2006). The PCR cassette for eachdeletion/integration was made by combining four fragments, A-B-U-C,either by overlapping PCR or by cloning the individual fragments, andgene to be integrated, into a plasmid prior to amplifying the entirecassette by PCR for the deletion/integration procedure. The PCR cassettecontained a selectable/counter-selectable marker, URA3 (Fragment U),consisting of the native CEN.PK 113-7D URA3 gene, along with thepromoter (250 bp upstream of the URA3 gene) and terminator (150 bpdownstream of the URA3 gene) regions. Fragments A (150 bp to 500 bplong) and C (250 bp long) corresponded to the sequence immediatelyupstream of the target gene (Fragment A) and the 3′ sequence of thetarget gene (Fragment C). Fragments A and C were used for integration ofthe cassette into the chromosome by homologous recombination. Fragment B(500 bp long) corresponded to the 500 bp immediately downstream of thetarget gene and was used for excision of the URA3 marker and Fragment Cfrom the chromosome by homologous recombination, as a direct repeat ofthe sequence corresponding to Fragment B was created upon integration ofthe cassette into the chromosome.

Using the PCR product ABUC cassette, the URA3 marker was firstintegrated into and then excised from the chromosome by homologousrecombination. The initial integration deleted the gene, excluding the3′ sequence. Upon excision, the 3′ region of the gene was also deleted.For integration of genes using this method, the gene to be integratedwas included in the cassette between fragments A and B.

FRA2 Deletion

The FRA2 deletion (also described in U.S. Appl. No. 61/380,563,incorporated by reference herein) was designed to delete 250 nucleotidesfrom the 3′ end of the coding sequence, leaving the first 113nucleotides of the FRA2 coding sequence intact. An in-frame stop codonwas present 7 nucleotides downstream of the deletion. The four fragmentsfor the PCR cassette for the scarless FRA2 deletion were amplified usingPhusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich,Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a GentraPuregene Yeast/Bact kit (Qiagen; Valencia, Calif.). FRA2 Fragment A wasamplified with primer oBP594 (SEQ ID NO: 961) and primer oBP595 (SEQ IDNO: 962), containing a 5′ tail with homology to the 5′ end of FRA2Fragment B. FRA2 Fragment B was amplified with primer oBP596 (SEQ ID NO:963), containing a 5′ tail with homology to the 3′ end of FRA2 FragmentA, and primer oBP597 (SEQ ID NO: 964), containing a 5′ tail withhomology to the 5′ end of FRA2 Fragment U. FRA2 Fragment U was amplifiedwith primer oBP598 (SEQ ID NO: 965), containing a 5′ tail with homologyto the 3′ end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO: 966),containing a 5′ tail with homology to the 5′ end of FRA2 Fragment C.FRA2 Fragment C was amplified with primer oBP600 (SEQ ID NO: 967),containing a 5′ tail with homology to the 3′ end of FRA2 Fragment U, andprimer oBP601 (SEQ ID NO: 968). PCR products were purified with a PCRPurification kit (Qiagen). FRA2 Fragment AB was created by overlappingPCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying withprimers oBP594 (SEQ ID NO: 961) and oBP597 (SEQ ID NO: 964). FRA2Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U andFRA2 Fragment C and amplifying with primers oBP598 (SEQ ID NO: 965) andoBP601 (SEQ ID NO: 968). The resulting PCR products were purified on anagarose gel followed by a Gel Extraction kit (Qiagen). The FRA2 ABUCcassette was created by overlapping PCR by mixing FRA2 Fragment AB andFRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO: 961) andoBP601 (SEQ ID NO: 968). The PCR product was purified with a PCRPurification kit (Qiagen).

Competent cells of PNY1503 were made and transformed with the FRA2 ABUCPCR cassette using a Frozen-EZ Yeast Transformation II kit (ZymoResearch; Orange, Calif.). Transformation mixtures were plated onsynthetic complete media lacking uracil supplemented with 1% ethanol at30° C. Transformants with a fra2 knockout were screened for by PCR withprimers oBP602 (SEQ ID NO: 969) and oBP603 (SEQ ID NO: 970) usinggenomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). Acorrect transformant was grown in YPE (yeast extract, peptone, 1%ethanol) and plated on synthetic complete medium supplemented with 1%ethanol and containing 5-fluoro-orotic acid (0.1%) at 30° C. to selectfor isolates that lost the URA3 marker. The deletion and marker removalwere confirmed by PCR with primers oBP602 (SEQ ID NO: 969) and oBP603(SEQ ID NO: 970) using genomic DNA prepared with a Gentra PuregeneYeast/Bact kit (Qiagen). The absence of the FRA2 gene from the isolatewas demonstrated by a negative PCR result using primers specific for thedeleted coding sequence of FRA2, oBP605 (SEQ ID NO: 971) and oBP606 (SEQID NO: 972). The correct isolate was selected as strain CEN.PK 113-7DMATa ura3A::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ and designated asPNY1505 (BP1135).

TABLE 16 Primers used in the FRA2 Deletion SEQ Primer ID Name NOPrimer Sequence oBP594 961 agctgtctcgtgttgtgggttt oBP595 962cttaataatagaacaatatcatcct ttacgggcatcttatagtgtcgtt oBP596 963gcgccaacgacactataagatgccc gtaaaggatgatattgttctatta oBP597 964tatggaccctgaaaccacagccaca ttgcaacgacgacaatgccaaacc oBP598 965tccttggtttggcattgtcgtcgtt gcaatgtggctgtggtttcagggt oBP599 966atcctctcgcggagtccctgttcag taaaggccatgaagctttttcttt oBP600 967attggaaagaaaaagcttcatggcc tttactgaacagggactccgcgag oBP601 968tcataccacaatcttagaccat oBP602 969 tgttcaaacccctaaccaacc oBP603 970tgttcccacaatctattaccta oBP605 971 tactgaacagggactccgcga oBP606 972tcataccacaatcttagacca

YPRCΔ15 Deletion and AFT1-L99A Integration

The YPRCΔ15 locus was deleted and replaced with AFT1-L99A along with thenative promoter region (800 bp) and terminator region (800 bp) fromAFT1. The scarless cassette for the YPRCΔ15 deletion-AFT1L99Aintegration was first cloned into plasmid pUC19-URA3MCS (described inU.S. Appl. No. 61/356,379, incorporated by reference herein). The vectoris pUC19 based and contains the sequence of the URA3 gene from S.cerevisiae CEN.PK 113-7D situated within a multiple cloning site (MCS).pUC19 (American Type Culture Collection, Manassas, Va.; ATCC #37254)contains the pMB1 replicon and a gene coding for beta-lactamase forreplication and selection in Escherichia coli. In addition to the codingsequence for URA3, the sequences from upstream (250 bp) and downstream(150 bp) of this gene are present for expression of the URA3 gene inyeast. The vector can be used for cloning purposes and can be used as ayeast integration vector.

The DNA encompassing the URA3 coding region along with 250 bp upstreamand 150 bp downstream of the URA3 coding region from Saccharomycescerevisiae CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures(CBS) Fungal Biodiversity Centre, Netherlands) genomic DNA was amplifiedwith primers oBP438 (SEQ ID NO: 1033), containing BamHI, AscI, PmeI, andFseI restriction sites, and oBP439 (SEQ ID NO: 1034), containing XbaI,Pad, and Nod restriction sites. Genomic DNA was prepared using a GentraPuregene Yeast/Bact kit (Qiagen). The PCR product and pUC19 were ligatedwith T4 DNA ligase after digestion with BamHI and XbaI to create vectorpUC19-URA3MCS. The vector was confirmed by PCR and sequencing withprimers oBP264 (SEQ ID NO:1031) and oBP265 (SEQ ID NO: 1032).

YPRCΔ15 Fragment A was amplified from genomic DNA, prepared as above,with primer oBP622 (SEQ ID NO: 973), containing a KpnI restriction site,and primer oBP623 (SEQ ID NO: 974), containing a 5′ tail with homologyto the 5′ end of YPRCΔ15 Fragment B. YPRCΔ15 Fragment B was amplifiedfrom genomic DNA with primer oBP624 (SEQ ID NO: 975), containing a 5′tail with homology to the 3′ end of YPRCΔ15 Fragment A, and primeroBP625 (SEQ ID NO: 976), containing a FseI restriction site. PCRproducts were purified with a PCR Purification kit (Qiagen). YPRCΔ15Fragment A-YPRCΔ15 Fragment B was created by overlapping PCR by mixingthe YPRCΔ15 Fragment A and YPRCΔ15 Fragment B PCR products andamplifying with primers oBP622 (SEQ ID NO: 973) and oBP625 (SEQ ID NO:976). The resulting PCR product was digested with KpnI and FseI andligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCSafter digestion with the appropriate enzymes. YPRCΔ15 Fragment C wasamplified from genomic DNA with primer oBP626 (SEQ ID NO: 977),containing a NotI restriction site, and primer oBP627 (SEQ ID NO: 978),containing a PacI restriction site. The YPRCΔ15 Fragment C PCR productwas digested with NotI and PacI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing YPRCΔ15 Fragments AB.AFT1-L99A, along with the native promoter region (800 bp) and terminatorregion (800 bp) from AFT1, was amplified using pRS411-AFT1(L99A)(described in Example 4 above) as template with primer oBP744 (SEQ IDNO: 979), containing an AscI restriction site, and primer oBP745 (SEQ IDNO: 980), containing a PmeI restriction site. The PCR product wasdigested with AscI and PmeI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing YPRCΔ15 Fragments ABC. Theentire integration cassette was amplified from the resulting plasmidwith primers oBP622 (SEQ ID NO: 973) and oBP627 (SEQ ID NO: 978).

Competent cells of PNY1503 were made and transformed with the YPRCΔ15deletion/AFT1-L99A integration cassette PCR product using a Frozen-EZYeast Transformation II kit (Zymo Research). Transformation mixtureswere plated on synthetic complete media lacking uracil supplemented with1% ethanol at 30° C. Transformants were grown in YPE (1% ethanol) andplated on synthetic complete medium supplemented with 1% EtOH andcontaining 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolatesthat lost the URA3 marker. The deletion of YPRCΔ15 and integration ofAFT1L99A were confirmed by PCR with external primers oBP636 (SEQ ID NO:981) and oBP637 (SEQ ID NO: 982) and with AFT1-L99A specific primerHY840 (SEQ ID NO: 983) and external primer oBP637 (SEQ ID NO: 982) usinggenomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen) andby colony PCR. Correct independent isolates of CEN.PK 113-7D MATaura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP yprcΔ15Δ::AFT1L99A weredesignated as strains PNY1541 and PNY1542.

TABLE 17 Primers used in the YPRCΔ15 Deletion and AFT1-L99A IntegrationSEQ Primer ID Name NO Primer Sequence oBP622 973aattggtaccccaaaaggaatattgggtcaga oBP623 974ccattgtttaaacggcgcgccggatcctttgc gaaaccctatgctctgt oBP624 975gcaaaggatccggcgcgccgtttaaacaatgg aaggtcgggatgagcat oBP625 976aattggccggcctacgtaacattctgtcaaccaa oBP626 977aattgcggccgcttcatatatgacgtaataaaat oBP627 978aattttaattaattttttttcttggaatcagtac oBP744 979aattggcgcgccagagtacaacgatcaccgcctg oBP745 980aattgtttaaacgaacgaaagttacaaaatctag oBP636 981 catttttttccctctaagaagcoBP637 982 tttttgcacagttaaactaccc HY840 983 CCAAAATCAGCCCCACGACGGCCATA

Example 13 Construction of Saccharomyces cerevisiae Strain BP1064(PNY1503)

The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,Netherlands) and contains deletions of the following genes: URA3, HIS3,PDC1, PDC5, PDC6, and GPD2.

Deletions, which completely removed the entire coding sequence, werecreated by homologous recombination with PCR fragments containingregions of homology upstream and downstream of the target gene andeither a G418 resistance marker or URA3 gene for selection oftransformants. The G418 resistance marker, flanked by loxP sites, wasremoved using Cre recombinase. The URA3 gene was removed by homologousrecombination to create a scarless deletion, or if flanked by loxP siteswas removed using Cre recombinase.

The scarless deletion procedure was adapted from Akada et al. 2006 Yeastv23 p399. In general, the PCR cassette for each scarless deletion wasmade by combining four fragments, A-B-U-C, by overlapping PCR. The PCRcassette contained a selectable/counter-selectable marker, URA3(Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, alongwith the promoter (250 bp upstream of the URA3 gene) and terminator (150bp downstream of the URA3 gene). Fragments A and C, each 500 bp long,corresponded to the 500 bp immediately upstream of the target gene(Fragment A) and the 3′ 500 bp of the target gene (Fragment C).Fragments A and C were used for integration of the cassette into thechromosome by homologous recombination. Fragment B (500 bp long)corresponded to the 500 bp immediately downstream of the target gene andwas used for excision of the URA3 marker and Fragment C from thechromosome by homologous recombination, as a direct repeat of thesequence corresponding to Fragment B was created upon integration of thecassette into the chromosome. Using the PCR product ABUC cassette, theURA3 marker was first integrated into and then excised from thechromosome by homologous recombination. The initial integration deletedthe gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp regionof the gene was also deleted. For integration of genes using thismethod, the gene to be integrated was included in the PCR cassettebetween fragments A and B.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxPcassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 986).pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and isflanked by loxP sites to allow recombination with Cre recombinase andremoval of the marker. PCR was done using Phusion DNA polymerase andprimers BK505 and BK506 (SEQ ID NOs: 987 and 988, respectively). TheURA3 portion of each primer was derived from the 5′ region upstream ofthe URA3 promoter and 3′ region downstream of the coding region suchthat integration of the loxP-kanMX-loxP marker resulted in replacementof the URA3 coding region. The PCR product was transformed into CEN.PK113-7D using standard genetic techniques (Methods in Yeast Genetics,2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.201-202) and transformants were selected on YPD containing G418 (100μg/ml) at 30 C. Transformants were screened to verify correctintegration by PCR using primers LA468 and LA492 (SEQ ID NOs: 989 and990, respectively) and designated CEN.PK 113-7D Δura3::kanMX.

HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template,prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia,Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO:991) and primer oBP453 (SEQ ID NO: 992), containing a 5′ tail withhomology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplifiedwith primer oBP454 (SEQ ID NO: 993), containing a 5′ tail with homologyto the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 994),containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U.HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 995),containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, andprimer oBP457 (SEQ ID NO: 996), containing a 5′ tail with homology tothe 5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primeroBP458 (SEQ ID NO: 997), containing a 5′ tail with homology to the 3′end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 998). PCR productswere purified with a PCR Purification kit (Qiagen). HIS3 Fragment AB wascreated by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment Band amplifying with primers oBP452 (SEQ ID NO: 991) and oBP455 (SEQ IDNO: 994). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQID NO: 995) and oBP459 (SEQ ID NO: 998). The resulting PCR products werepurified on an agarose gel followed by a Gel Extraction kit (Qiagen).The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQID NO: 991) and oBP459 (SEQ ID NO: 998). The PCR product was purifiedwith a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformedwith the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast TransformationII kit (Zymo Research; Orange, Calif.). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a his3 knockout were screened forby PCR with primers oBP460 (SEQ ID NO: 999) and oBP461 (SEQ ID NO: 1000)using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::kanMX Δhis3::URA3.

KanMX Marker Removal from the ΔUra3 Site and URA3 Marker Removal fromthe Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMXΔhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 1011, described in U.S.Provisional Application No. 61/290,639) using a Frozen-EZ YeastTransformation II kit (Zymo Research) and plating on synthetic completemedium lacking histidine and uracil supplemented with 2% glucose at 30°C. Transformants were grown in YP supplemented with 1% galactose at 30°C. for ˜6 hours to induce the Cre recombinase and KanMX marker excisionand plated onto YPD (2% glucose) plates at 30° C. for recovery. Anisolate was grown overnight in YPD and plated on synthetic completemedium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select forisolates that lost the URA3 marker. 5-FOA resistant isolates were grownin and plated on YPD for removal of the pRS423::P_(GAL1)-cre plasmid.Isolates were checked for loss of the KanMX marker, URA3 marker, andpRS423::P_(GAL1)-cre plasmid by assaying growth on YPD+G418 plates,synthetic complete medium lacking uracil plates, and synthetic completemedium lacking histidine plates. A correct isolate that was sensitive toG418 and auxotrophic for uracil and histidine was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletionsand marker removal were confirmed by PCR and sequencing with primersoBP450 (SEQ ID NO: 1001) and oBP451 (SEQ ID NO: 1002) for Δura3 andprimers oBP460 (SEQ ID NO: 999) and oBP461 (SEQ ID NO: 1000) for Δhis3using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen).

PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with aGentra Puregene Yeast/Bact kit (Qiagen). PDC6 Fragment A was amplifiedwith primer oBP440 (SEQ ID NO: 1003) and primer oBP441 (SEQ ID NO:1004), containing a 5′ tail with homology to the 5′ end of PDC6 FragmentB. PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 1005),containing a 5′ tail with homology to the 3′ end of PDC6 Fragment A, andprimer oBP443 (SEQ ID NO: 1006), containing a 5′ tail with homology tothe 5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primeroBP444 (SEQ ID NO: 1007), containing a 5′ tail with homology to the 3′end of PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 1008), containinga 5′ tail with homology to the 5′ end of PDC6 Fragment C. PDC6 FragmentC was amplified with primer oBP446 (SEQ ID NO: 1009), containing a 5′tail with homology to the 3′ end of PDC6 Fragment U, and primer oBP447(SEQ ID NO: 1010). PCR products were purified with a PCR Purificationkit (Qiagen). PDC6 Fragment AB was created by overlapping PCR by mixingPDC6 Fragment A and PDC6 Fragment B and amplifying with primers oBP440(SEQ ID NO: 1003) and oBP443 (SEQ ID NO: 1006). PDC6 Fragment UC wascreated by overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment Cand amplifying with primers oBP444 (SEQ ID NO: 1007) and oBP447 (SEQ IDNO: 1010). The resulting PCR products were purified on an agarose gelfollowed by a Gel Extraction kit (Qiagen). The PDC6 ABUC cassette wascreated by overlapping PCR by mixing PDC6 Fragment AB and PDC6 FragmentUC and amplifying with primers oBP440 (SEQ ID NO: 1003) and oBP447 (SEQID NO: 1010). The PCR product was purified with a PCR Purification kit(Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made andtransformed with the PDC6 ABUC PCR cassette using a Frozen-EZ YeastTransformation II kit (Zymo Research). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a pdc6 knockout were screened forby PCR with primers oBP448 (SEQ ID NO: 1012) and oBP449 (SEQ ID NO:1013) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::loxP Δhis3 Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPDand plated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR and sequencing withprimers oBP448 (SEQ ID NO: 1012) and oBP449 (SEQ ID NO: 1013) usinggenomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). Theabsence of the PDC6 gene from the isolate was demonstrated by a negativePCR result using primers specific for the coding sequence of PDC6,oBP554 (SEQ ID NO: 1014) and oBP555 (SEQ ID NO: 1015). The correctisolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 anddesignated as BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region fromStreptococcus mutans ATCC #700610. The A fragment followed by the ilvDcoding region from Streptococcus mutans for the PCR cassette for thePDC1 deletion-ilvDSm integration was amplified using Phusion HighFidelity PCR Master Mix (New England BioLabs) and NYLA83 (described inU.S. Provisional Application No. 61/246,709) genomic DNA as template,prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 FragmentA-ilvDSm (SEQ ID NO: 1053) was amplified with primer oBP513 (SEQ ID NO:1016) and primer oBP515 (SEQ ID NO: 1017), containing a 5′ tail withhomology to the 5′ end of PDC1 Fragment B. The B, U, and C fragments forthe PCR cassette for the PDC1 deletion-ilvDSm integration were amplifiedusing Phusion High Fidelity PCR Master Mix (New England BioLabs) andCEN.PK 113-7D genomic DNA as template, prepared with a Gentra PuregeneYeast/Bact kit (Qiagen). PDC1 Fragment B was amplified with primeroBP516 (SEQ ID NO: 1018) containing a 5′ tail with homology to the 3′end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 1019),containing a 5′ tail with homology to the 5′ end of PDC1 Fragment U.PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO: 1020),containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B, andprimer oBP519 (SEQ ID NO: 1021), containing a 5′ tail with homology tothe 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primeroBP520 (SEQ ID NO: 1022), containing a 5′ tail with homology to the 3′end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 1023). PCRproducts were purified with a PCR Purification kit (Qiagen). PDC1Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513(SEQ ID NO: 1016) and oBP517 (SEQ ID NO: 1019). PDC1 Fragment UC wascreated by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment Cand amplifying with primers oBP518 (SEQ ID NO: 1020) and oBP521 (SEQ IDNO: 1023). The resulting PCR products were purified on an agarose gelfollowed by a Gel Extraction kit (Qiagen). The PDC1 A-ilvDSm-BUCcassette (SEQ ID NO: 1054) was created by overlapping PCR by mixing PDC1Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying with primersoBP513 (SEQ ID NO: 1016) and oBP521 (SEQ ID NO: 1023). The PCR productwas purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made andtransformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZYeast Transformation II kit (Zymo Research). Transformation mixtureswere plated on synthetic complete media lacking uracil supplemented with2% glucose at 30° C. Transformants with a pdc1 knockout ilvDSmintegration were screened for by PCR with primers oBP511 (SEQ ID NO:1024) and oBP512 (SEQ ID NO: 1025) using genomic DNA prepared with aGentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC1 genefrom the isolate was demonstrated by a negative PCR result using primersspecific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 1026) andoBP551 (SEQ ID NO: 1027). A correct transformant was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grownovernight in YPD and plated on synthetic complete medium containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC1, integration of ilvDSm, and markerremoval were confirmed by PCR and sequencing with primers oBP511 (SEQ IDNO: 1024) and oBP512 (SEQ ID NO: 1025) using genomic DNA prepared with aGentra Puregene Yeast/Bact kit (Qiagen). The correct isolate wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmand designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region fromAchromobacter xylosoxidans. A segment of the PCR cassette for the PDC5deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.

pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 genefrom Saccharomyces cerevisiae situated within a multiple cloning site(MCS). pUC19 contains the pMB1 replicon and a gene coding forbeta-lactamase for replication and selection in Escherichia coli. Inaddition to the coding sequence for URA3, the sequences from upstreamand downstream of this gene were included for expression of the URA3gene in yeast. The vector can be used for cloning purposes and can beused as a yeast integration vector.

The DNA encompassing the URA3 coding region along with 250 bp upstreamand 150 bp downstream of the URA3 coding region from Saccharomycescerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438(SEQ ID NO: 1033), containing BamHI, AscI, PmeI, and FseI restrictionsites, and oBP439 (SEQ ID NO: 1034), containing XbaI, Pad, and NotIrestriction sites, using Phusion High-Fidelity PCR Master Mix (NewEngland BioLabs). Genomic DNA was prepared using a Gentra PuregeneYeast/Bact kit (Qiagen). The PCR product and pUC19 (SEQ ID NO: 1056)were ligated with T4 DNA ligase after digestion with BamHI and XbaI tocreate vector pUC19-URA3MCS. The vector was confirmed by PCR andsequencing with primers oBP264 (SEQ ID NO: 1031) and oBP265 (SEQ ID NO:1032).

The coding sequence of sadB and PDC5 Fragment B were cloned intopUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCRcassette. The coding sequence of sadB was amplified using pLH468-sadB(SEQ ID NO: 1051) as template with primer oBP530 (SEQ ID NO: 1035),containing an AscI restriction site, and primer oBP531 (SEQ ID NO:1036), containing a 5′ tail with homology to the 5′ end of PDC5 FragmentB. PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 1037),containing a 5′ tail with homology to the 3′ end of sadB, and primeroBP533 (SEQ ID NO: 1038), containing a PmeI restriction site. PCRproducts were purified with a PCR Purification kit (Qiagen). sadB-PDC5Fragment B was created by overlapping PCR by mixing the sadB and PDC5Fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO:1035) and oBP533 (SEQ ID NO: 1038). The resulting PCR product wasdigested with AscI and PmeI and ligated with T4 DNA ligase into thecorresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. The resulting plasmid was used as a template foramplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ IDNO: 1039) and oBP546 (SEQ ID NO: 1040), containing a 5′ tail withhomology to the 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplifiedwith primer oBP547 (SEQ ID NO: 1041) containing a 5′ tail with homologyto the 3′ end of PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQID NO: 1042). PCR products were purified with a PCR Purification kit(Qiagen). PDC5 sadB-Fragment B-Fragment U-Fragment C was created byoverlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U and PDC5Fragment C and amplifying with primers oBP536 (SEQ ID NO: 1039) andoBP539 (SEQ ID NO: 1042). The resulting PCR product was purified on anagarose gel followed by a Gel Extraction kit (Qiagen). The PDC5A-sadB-BUC cassette (SEQ ID NO: 1055) was created by amplifying PDC5sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ ID NO:1043), containing a 5′ tail with homology to the 50 nucleotidesimmediately upstream of the native PDC5 coding sequence, and oBP539 (SEQID NO: 1042). The PCR product was purified with a PCR Purification kit(Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmwere made and transformed with the PDC5 A-sadB-BUC PCR cassette using aFrozen-EZ Yeast Transformation II kit (Zymo Research). Transformationmixtures were plated on synthetic complete media lacking uracilsupplemented with 1% ethanol (no glucose) at 30° C. Transformants with apdc5 knockout sadB integration were screened for by PCR with primersoBP540 (SEQ ID NO: 1044) and oBP541 (SEQ ID NO: 1045) using genomic DNAprepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence ofthe PDC5 gene from the isolate was demonstrated by a negative PCR resultusing primers specific for the coding sequence of PDC5, oBP552 (SEQ IDNO: 1046) and oBP553 (SEQ ID NO: 1047). A correct transformant wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmΔpdc5::sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 wasgrown overnight in YPE (1% ethanol) and plated on synthetic completemedium supplemented with ethanol (no glucose) and containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC5, integration of sadB, and markerremoval were confirmed by PCR with primers oBP540 (SEQ ID NO: 1044) andoBP541 (SEQ ID NO: 1045) using genomic DNA prepared with a GentraPuregene Yeast/Bact kit (Qiagen). The correct isolate was selected asstrain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadBand designated as BP913.

GPD2 Deletion

To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxPcassette (SEQ ID NO: 1057) was PCR-amplified using loxP-URA3-loxP PCR(SEQ ID NO: 1052) as template DNA. loxP-URA3-loxP contains the URA3marker from (ATCC #77107) flanked by loxP recombinase sites. PCR wasdone using Phusion DNA polymerase and primers LA512 and LA513 (SEQ IDNOs: 1029 and 1030, respectively). The GPD2 portion of each primer wasderived from the 5′ region upstream of the GPD2 coding region and 3′region downstream of the coding region such that integration of theloxP-URA3-loxP marker resulted in replacement of the GPD2 coding region.The PCR product was transformed into BP913 and transformants wereselected on synthetic complete media lacking uracil supplemented with 1%ethanol (no glucose). Transformants were screened to verify correctintegration by PCR using primers oBP582 and AA270 (SEQ ID NOs: 1048 and1049, respectively).

The URA3 marker was recycled by transformation with pRS423::P_(GAL1)-cre(SEQ ID NO: 1011) and plating on synthetic complete media lackinghistidine supplemented with 1% ethanol at 30° C. Transformants werestreaked on synthetic complete medium supplemented with 1% ethanol andcontaining 5-fluoro-orotic acid (0.1%) and incubated at 30 C to selectfor isolates that lost the URA3 marker. 5-FOA resistant isolates weregrown in YPE (1% ethanol) for removal of the pRS423::P_(GAL1)-creplasmid. The deletion and marker removal were confirmed by PCR withprimers oBP582 (SEQ ID NO: 1048) and oBP591 (SEQ ID NO: 1050). Thecorrect isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as BP1064.

Example 14 Shake Flask Experiment to Measure 2,3-dihydroxyisovalerateAccumulation and Isobutanol Production

The purpose of this Example was to show the effect on accumulation ofthe isobutanol pathway intermediate 2,3-dihydroxyisovalerate (DHIV) andshow isobutanol production in isobutanologen strains with an integratedcopy of the AFT1-L99A gene or a FRA2 deletion compared to the parentstrain. Strains were transformed with isobutanol pathway plasmids pYZ090(SEQ ID NO: 984; described in U.S. patent application Ser. No.61/368,436, incorporated by reference herein) and pLH468 (SEQ ID NO:985; described in U.S. application Ser. No. 61/246,844, incorporated byreference herein). These plasmids are also described briefly, asfollows.

pYZ090 (SEQ ID NO: 984) was constructed to contain a chimeric genehaving the coding region of the alsS gene from Bacillus subtilis (ntposition 457-2172) expressed from the yeast CUP1 promoter (nt 2-449) andfollowed by the CYC1 terminator (nt 2181-2430) for expression of ALS,and a chimeric gene having the coding region of the ilvC gene fromLactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter(2433-3626) and followed by the ILV5 terminator (nt 4682-5304) forexpression of KARI.

pLH468 (SEQ ID NO: 985) was constructed to contain: a chimeric genehaving the coding region of the ilvD gene from Streptococcus mutans (ntposition 3313-4849) expressed from the S. cerevisiae FBA1 promoter (nt2109-3105) followed by the FBA1 terminator (nt 4858-5857) for expressionof DHAD; a chimeric gene having the coding region of codon optimizedhorse liver alcohol dehydrogenase (nt 6286-7413) expressed from the S.cerevisiae GPM1 promoter (nt 7425-8181) followed by the ADH1 terminator(nt 5962-6277) for expression of ADH; and a chimeric gene having thecoding region of the codon-optimized kivD gene from Lactococcus lactis(nt 9249-10895) expressed from the TDH3 promoter (nt 10896-11918)followed by the TDH3 terminator (nt 8237-9235) for expression of KivD.

A transformant of PNY1503 (parent strain) was designated PNY1504. Atransformant of PNY1505 (fra2 deletion strain) was designated PNY1506.Transformants of PNY1541 and PNY1542 (AFT1-L99A integration strains)were designated PNY1543 and PNY1544, for PNY1541, and PNY1545 andPNY1546, for PNY1542.

Strains were grown in synthetic medium (Yeast Nitrogen Base withoutAmino Acids (Sigma-Aldrich, St. Louis, Mo.) and Yeast Synthetic Drop-OutMedia Supplement without uracil and histidine (Clontech, Mountain View,Calif.)) supplemented with 100 mM MES pH5.5, 0.2% glucose, and 0.2%ethanol. Overnight cultures were grown in 15 mL of medium in 125 mLvented Erlenmeyer flasks at 30° C., 225 RPM in a New BrunswickScientific I24 shaker. 18 ml of medium in 125 mL tightly-cappedErlenmeyer flasks was inoculated with overnight culture to an OD₆₀₀ 0.5and grown for six hours at 30° C., 225 RPM in a New Brunswick Scientific124 shaker. After six hours, glucose was added to 2.5%, yeast extractwas added to 5 g/L, and peptone was added to 10 g/L (time 0 hours).After 24 and 48 hours, culture supernatants (collected using Spin-Xcentrifuge tube filter units, Costar Cat. No. 8169) were analyzed byHPLC (method described in U.S. Patent Appl. Pub. No. US 2007/0092957,incorporated by reference herein) and LC/MS. Glucose and isobutanolconcentrations were determined by HPLC. DHIV was separated andquantified by LC/MS on a Waters (Milford, Mass.) AcquityTQD system,using an Atlantis T3 (part #186003539) column. The column was maintainedat 30° C. and the flow rate was 0.5 mL/min. The A mobile phase was 0.1%formic acid in water, and the B mobile phase was 0.1% formic acid inacetonitrile. Each run consisted of 1 min at 99% A, a linear gradientover 1 min to 25% B, followed by 1 min at 99% A. The column effluent wasmonitored for peaks at m/z=133 (negative ESI), with cone voltage 32.5V,by Waters ACQ_TQD (s/n QBA688) mass spectometry detector. DHIV typicallyemerged at 1.2 min. Baseline separation was obtained and peak areas forDHIV were converted to μM DHIV concentrations by reference to analysesof standards solutions made from a 1 M aqueous stock.

Table 18 shows the DHIV molar yield (moles of DHIV per moles of glucoseconsumed) and isobutanol titer of the AFT1-L99A strains (PNY1543,PNY1544, PNY1545, and PNY1546) and the FRA2 deletion strain (PNY1506)compared to the parent strain background (PNY1504) at 24 and 48 hours.AFT1-L99A expression or the FRA2 deletion both led to approximately a50% decrease in the accumulation of DHIV.

TABLE 18 DHIV molar yield and isobutanol titer. 24 Hr 48 Hr 24 Hr 48 HrDHIV Yield DHIV Yield Isobutanol Isobutanol Strain (mol/mol) (mol/mol)Titer (g/L) Titer (g/L) PNY1504 0.044 0.035 3.7 4.2 PNY1543- 0.017 0.0154.1 5.8 PNY1544 PNY1545- 0.019 0.018 4.6 5.5 PNY1546 PNY1506 0.022 0.0203.8 4.7

Data are the average of two independent flasks, for PNY1504 and PNY1506,and two independent transformants for the AFT1-L99A strains(PNY1543-PNY1544 and PNY1545-PNY1546).

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

TABLE 12 HMMER2.0 [2.2g] Program name and version NAME dhad_for_hmm Nameof the input sequence alignment file LENG 564 Length of the alignment:include indels ALPH Amino Type of residues MAP yes Map of the matchstates to the columns of the alignmentCOM/app/public/hmmer/current/bin/hmmbuild -F dhad-exp_hmmdhad_for_hmm.aln Commands used to generate the file: this one means thathmmbuild (default patrameters) was applied to the alignment fileCOM/app/public/hmmer/current/bin/hmmcalibrate dhad-exp_hmm Commands usedto generate the file: this one means that hmmcalibrate (defaultparametrs) was applied to the hmm profile NSEQ 8 Number of sequences inthe alignment file DATE Tue Jun 3 10:48:24 2008 When was the filegenerated XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4 NULT −4 −8455 Thetransition probability distribution for the null model (single G state).NULE 595 −1558 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45531 201 The symbol emission probability distribution for the null model(G state); consists of K (e.g. 4 or 20) integers. The null 384 −1998−644 probability used to convert these back to model probabilities is1/K. EVD −499.650970 0.086142 The extreme value distribution parametersμ and lambda respectively; both floating point values. Lambda ispositive and nonzero. These values are set when the model is calibratedwith hmmcalibrate. A C D E F G H I K Position in HMM m->m m->i m->d i->mi->i d->m d->d b->m m->e L M N P Q R S T V W Y alignment −538 * −1684 1(M) −233 −1296 99 1223 −1477 −1132 89 −1122 420 −1248 1757 1553 −1296464 −24 −190 −188 −838 −1578 −985 6 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −29 −6203 −7245−894 −1115 −701 −1378 −538 *  2(E) −220 −1288 232 1356 −1807 1016 −70−1474 190 −1584 −775 132 −1298 300 −282 −183 1140 −1092 −1872 −1262 7 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −29 −6203 −7245 −894 −1115 −701 −1378 * *  3(K) −448−1932 1558 658 −2220 −1048 40 −1983 1569 −1938 −1091 1558 −1319 450 −193−278 −419 −1552 −2121 −1397 8 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −29 −6203 −7245 −894−1115 −701 −1378 * *  4(V) −404 −498 −1497 −939 −588 −1810 −640 1591 914−127 335 −962 −1866 −562 −767 −868 −357 1720 −1169 −763 9 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −29 −6203 −7245 −894 −1115 −701 −1378 * *  5(E) −265 −1340 −521376 −1572 −1189 113 −1125 1345 −1287 −496 99 −1321 505 198 −218 −205597 −1598 −1032 10 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −29 −6203 −7245 −894 −1115 −701−1378 * *  6(S) 256 −397 −1014 −830 −1841 −646 −862 −1443 −767 −1740−963 −568 −1249 −651 −1007 2267 1586 −862 −2080 −1672 11 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −29 −6203 −7245 −894 −1115 −701 −1378 * *  7(M) −990 −889 −2630 157−513 −2514 −1346 1309 −1767 820 3683 −1898 −2491 −1496 −1799 −1589 −925150 −1336 −1041 12 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  8(E) 588 −1875 −194 1536 −2188 −1373 −59 −1931 957 −1890 −977904 292 393 −162 483 −372 −1495 −2070 −1391 13 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  9(N) −514 −1116 1207 −315 447−1650 −304 −778 −224 825 −277 1457 −1738 −123 −618 −627 −454 −603 −1186763 14 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 10(N) −815 −1190 −1360 −922 −904 −1967 −797 −442 −670 381 1700 3009−2099 −654 −934 −1051 −791 −445 −1490 −979 15 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  11(K) −1530 −2498 −1722 −855−3141 −2246 −428 −2627 2828 −2404 −1656 −927 662 −2 2047 −1421 −1337−2324 −2357 −2081 16 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  12(Y) −872 −1887 −861 −290 −1369 −1801 1662 −1797 325 −1793−1031 893 −1876 56 2219 −812 −780 −1514 −1565 2287 17 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * *  13(S) −830 −1586 −1471 −1099−2717 −1642 −1010 −2479 −266 −2518 −1746 −1065 −2069 −676 1822 2748−1000 −1950 −2597 −2189 18 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  14(Q) −851 −2131 −775 −153 −2554 −1735 −211 −2205 1908−2094 −1244 −386 −1802 2254 974 1001 −747 −1819 −2181 −1667 19 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  15(T) −405 −1258−618 −100 −1490 −1466 1158 −1121 1 −1299 −514 578 −1607 65 −433 960 1849343 −1677 −1143 20 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  16(I) −1772 −1325 −4307 −3877 −1405 −3993 −3383 2935 −3705820 −217 −3632 −3761 −3400 −3682 −3260 −1742 2033 −2838 −2525 21 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  17(T) −1018 −1329−2004 −1771 −409 −1993 −1000 −1256 −1512 −1464 −966 −1543 −2367 −1428−1638 −1257 3050 −1090 −1012 2448 22 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  18(Q) −1509 −3056 1970 44 −3310 −1666−896 −3242 −877 −3158 −2439 −322 −2123 3562 −1493 −1259 −1550 −2779−3260 −2446 23 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  19(D) −1006 −2199 2178 −88 −3159 1997 −936 −2974 −948 −2977−2174 −382 −1960 −589 −1571 1295 −1157 −2369 −3178 −2430 24 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  20(M) 445 −796 −1082−521 −841 −1643 −412 −403 −370 −692 2213 −646 536 1166 −698 −630 660 831−1204 −767 25 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 39445 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 21(Q) 741 −990 −1025 −507 −1249 −1551 −519 −720 −357 −1062 −345 −635−1739 1770 −713 −589 1576 1129 −1559 −1097 26 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  22(R) −1753 −2648 −2072 −1047−3365 −2405 −452 −2782 1989 −2495 −1773 −1062 −2379 2402 2643 −1629−1506 −2504 −2397 −2190 27 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  23(S) −330 −1010 −1820 −1628 −2778 −1229 −1652 −2481−1592 −2691 −1841 −1273 2130 −1426 −1834 2449 1034 −1716 −2961 −2594 28— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  24(P) 1882−1119 −2231 −2302 −3062 −1360 −2209 −2710 −2339 −3013 −2243 −1676 3304−2117 −2409 −742 −918 −1916 −3263 −3022 29 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  25(N) 969 −1230 −1066 −915 −2593 −1313−1196 −2242 −1033 −2447 −1626 3197 −1850 −898 −1392 −582 1155 −1644−2736 −2256 30 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  26(R) −1847 −2640 −2014 −1161 −3282 −2428 −579 −2818 687−2553 −1869 −1165 −2462 2447 3181 −1746 −1630 −2555 −2447 −2228 31 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  27(A) 3048−932 −2480 −2533 −3075 −1200 −2274 −2765 −2501 −3071 −2221 −1658 −1948−2205 −2512 1225 −739 −1842 −3322 −3078 32 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  28(M) −2406 −2296 −3638 −3594 −1525−3105 −2824 −1047 −3121 −596 5043 −3293 −3425 −3046 −2996 −2911 −2552−1398 −2513 −2207 33 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  29(Y) −1674 −1506 −2863 −2464 596 −2872 2251 −972 −2024 2197−552 −1986 −2876 −1739 −1988 −1987 −1601 −1002 −95 2332 34 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  30(Y) −2013 −2305−2428 −1781 −328 −2709 −654 −2240 −258 −2064 −1626 −1631 −2788 −899 2789−2017 −1896 −2130 −857 3434 35 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  31(A) 2822 −1031 −2418 −2539 −3226 1898 −2364−2941 −2626 −3229 −2379 −1722 −2026 −2302 −2634 −654 −848 −1983 −3415−3226 36 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 32(I) −1247 −941 −3569 −3039 −1082 −3101 −2185 2227 −2763 766 −76 −2700−3050 −2469 −2697 −2253 1322 1974 −1988 −1633 37 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  33(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 38 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  34(F) −1511 −1236 −3511 −3017 2747 −2982 −1069 −260−2651 992 2737 −2407 −2904 −2088 −2418 −2099 −1434 −489 −537 2056 39 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  35(Q) −576−1869 −401 92 −2232 831 −173 −1930 1505 −1913 −1042 −186 −1620 1653 −51−482 1346 −1534 −2098 −1490 40 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  36(D) −1352 −3066 3028 1349 −3303 −1566 −724 −31411155 −3043 −2267 −165 −1991 −354 −1350 −1086 −1368 −2659 −3221 −2356 41— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  37(E) −1507−3288 2042 2762 −3520 515 −853 −3401 −981 −3296 −2566 −182 −2064 −503−1753 −1209 −1553 −2895 −3486 −2547 42 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  38(D) −1445 −2778 3529 −53 −3524 −1590−1129 −3476 −1367 −3459 −2774 −396 −2156 −825 −2122 554 −1609 −2880−3582 −2717 43 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  39(F) −2658 −2176 −4213 −4000 3815 −3933 −1352 −531 −36381121 −19 −3184 −3709 −2820 −3296 −3219 −2579 −1037 −601 403 44 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  40(D) −684 −21931738 1460 −2494 −1437 −249 −2257 1694 −2199 −1308 −62 −1637 185 −450−531 633 −1808 −2374 −1657 45 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  41(K) −2620 −2961 −2461 −2046 −3743 −2791 −1570−3603 3784 −3387 −2839 −2048 −3039 −1260 −465 −2604 −2536 −3331 −3001−2988 46 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 42(P) 1882 −1119 −2231 −2302 −3062 −1360 −2209 −2710 −2339 −3013 −2243−1676 3304 −2117 −2409 −742 −918 −1916 −3263 −3022 47 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * *  43(I) −1006 −992 −2347 −1784−650 −2452 −1256 2372 −1386 77 2213 −1720 −2455 2030 −1490 −1528 −946106 −1441 −1111 48 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  44(V) −1771 −1603 −3750 −3689 −2037 −3050 −3231 403 −3479−1154 −1076 −3246 −3399 −3383 −3437 −2628 −1917 3536 −3074 −2677 49 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  45(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 50 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  46(I) −1759 −1303 −4330 −3968 −1751−4051 −3743 3027 −3837 −597 −528 −3729 −3875 −3688 −3910 −3369 −17512438 −3259 −2819 51 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  47(V) 1736 −1012 −3546 −3078 −1377 −3073 −2434 2052 −2843−608 −331 −2754 −3122 −2619 −2855 −2270 −1277 2193 −2333 −1941 52 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  48(N) −686 −1511−702 −806 −2927 −1386 −1339 −2841 −1264 −2950 −2137 2702 −1979 −1062−1648 2444 −971 −2105 −3054 −2475 53 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  49(M) −411 −857 −1800 −1434 −1528 1914−1202 −1029 −1247 −1347 2989 −1217 −1912 −1119 −1444 −676 1550 −767−1922 −1539 54 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  50(W) −782 −1258 793 −683 1193 346 2051 −932 −556 −1092 −441−798 −1993 −426 −909 −904 −720 −779 3163 1546 55 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  51(W) 1009 −798 −1470 −935 −463−1773 −545 −460 −751 −736 −66 −943 −1904 −606 −1002 1604 −507 −322 25351521 56 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 52(D) −1137 −2711 2125 1647 −2995 −1523 −617 −2786 −528 −2743 −1933−150 −1897 −234 −1165 −924 2117 −2331 −2948 −2141 57 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * *  53(I) −599 −1102 −1031 −829−1522 1429 −927 2119 −880 −1369 −699 1692 −1938 −759 −1188 −799 −698−689 −1887 −1419 58 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  54(T) −666 −1412 −954 −984 −2702 −1428 −1357 −2418 −1208−2650 −1886 2293 −2000 −1101 −1519 −787 2967 −1835 −2866 −2360 59 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  55(P) −632 −1230−2074 −2144 −2996 −1453 −2116 −2631 −2128 −2928 −2213 −1658 3610 −2006−2221 −852 1302 −1931 −3185 −2917 60 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  56(C) −2476 5735 −4102 −4358 −3712−2763 −3545 −3518 −4167 −3859 −3569 −3631 −3363 −4030 −3832 −2793 −2860−3158 −3464 −3718 61 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  57(N) −2171 −2655 −1458 −1748 −3334 −2364 −2267 −3943 −2365−3936 −3437 4205 −2932 −2205 −2608 −2224 −2439 −3392 −3253 −2909 62 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  58(M) 672−918 −3119 −2578 −742 −2668 −1734 1807 −2263 16 3713 −2271 −2704 −1960−2216 −1806 −1058 493 −1612 −1306 63 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  59(H) −1525 −2164 −1235 −1346 −25092296 4235 −3172 −1516 −3178 −2523 −1448 −2541 −1520 −1760 −1591 −1741−2656 −2681 −2065 64 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  60(L) −2478 −2009 −4717 −4196 −568 −4424 −3262 1334 −38872824 604 −4085 −3872 −3088 −3590 −3717 −2380 −199 −2217 −2207 65 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  61(H) −682 −21911015 275 −2485 396 2379 −2251 62 −2197 −1307 1826 −1636 1527 −480 −529−641 −1803 −2375 −1654 66 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  62(D) −575 −1920 1979 184 −2299 94 −242 −2029 114 −2023−1144 −120 −1608 186 1063 −469 1413 −1605 −2229 −1561 67 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * *  63(L) −2618 −2139 −4597−4163 2144 −4285 −2334 −83 −3854 2690 538 −3771 −3806 −2950 −3488 −3563−2505 −751 −1442 −808 68 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  64(A) 2657 −1033 −2408 −2532 −3233 2193 −2364 −2950−2626 −3237 −2386 −1719 −2027 −2301 −2635 −655 −850 −1988 −3420 −3231 69— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  65(K) −443−1857 958 270 −2158 −1393 −66 −1890 1839 −442 −957 −36 −1499 1204 −132616 −382 −1469 −2048 −1383 70 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  66(C) 605 1553 739 −17 −1374 −1488 −182 260 969−203 −397 −263 −1573 159 691 −426 −331 −761 −1567 −1032 71 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  67(A) 2327 −956 −3193−2728 −1289 −2677 −2114 1664 −2485 −601 −288 −2403 −2839 −2263 −2523−1871 −1126 1617 −2143 −1765 72 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  68(K) −532 −1656 −490 1321 −1891 −1527 −172 −1242206 −1591 −782 −223 −1619 237 −106 −482 −464 −98 −1904 −1326 73 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  69(H) 384 −1854936 889 −2165 −1363 1498 −1909 1111 −1866 −948 1091 −1464 421 −131 −284−342 −69 −2043 −1364 74 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  70(G) 1823 −932 −2330 −2313 −3120 2511 −2158 −2865 −2331−3098 −2209 −1563 −1912 −2032 −2419 1138 −706 −1883 −3328 −3077 75 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  71(V) −1760−1333 −4244 −3789 −1262 −3902 −3190 1495 −3588 1270 −96 −3536 −3677−3238 −3534 −3148 −1725 2865 −2654 −2373 76 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  72(W) −1054 −2172 −1112 −403 −2566−1917 −286 −2196 2516 −2095 −1292 1183 −1958 140 1333 −959 −922 −18672591 −1720 77 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 39445 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 73(D) 611 −1995 1525 937 −2295 −1400 −148 −2043 211 −2006 −1106 −37−1553 1420 −312 −408 1235 −1609 −2193 −1499 78 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  74(A) 2716 −902 −2380 −2205 −2799−1197 −1975 −2459 −2081 −2736 −1895 −1520 −1895 −1844 −2201 1191 1299−1669 −3045 −2758 79 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  75(G) −1709 −2833 2424 −409 −3781 2819 −1457 −3777 −1728−3733 −3076 −739 −2389 −1180 −2441 −1557 −1893 −3158 −3660 −3038 80 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −212 −2909 −8150 −273 −2534 −701 −1378 * *  76(A) 2529−1119 −2614 −2330 −1245 −1983 −1829 −377 −2042 1435 −341 −1937 −2411−1873 −2088 −1266 −1059 −397 −2063 −1713 82 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  77(W) −472 −361 −2421 −1812 −298 −1979−826 1164 −1486 −143 2485 873 −2028 −1185 −1426 −1048 −412 1116 2999−454 83 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 78(P) −1198 −1737 −2187 −2394 −3665 2006 −2550 −3630 −2743 −3756 −3008−2052 3474 −2495 −2835 −1401 −1593 −2736 −3511 −3519 84 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * *  79(Q) −999 −1075 −2106−1568 −726 −2370 −1175 83 −1185 1373 218 −1566 −2400 2445 −1340 −1445−946 1441 −1501 −1146 85 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  80(Q) −885 −779 −2609 −2018 −481 −2414 −1253 1645 −1736799 1924 −1827 −2405 2262 −1752 −1484 −821 802 −1240 −935 86 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  81(F) −3342 −2776−4026 −4232 4354 −3545 −1431 −2315 −4038 −1801 −1900 −3299 −3780 −3350−3645 −3490 −3420 −2566 −739 349 87 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * *  82(G) −998 −2100 −120 −175 −2567 2528 2174−2558 −587 −2583 −1806 1422 −1966 −461 −1038 −925 −1088 −2095 −2657−1948 88 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 83(T) −1213 −1674 −2755 −2906 −3163 −1922 −2659 −2698 −2788 −3105 −2612−2311 −2600 −2708 −2753 −1463 3819 −2197 −3286 −3156 89 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * *  84(I) −1286 −1279 −2907−2683 −1446 −2549 −2198 3290 −2407 −726 −534 −2386 1172 −2299 −2437−1895 −1392 283 −2302 −1913 90 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  85(T) −493 −1105 −2189 −2267 −3101 1880 −2196−2791 −2334 −3081 −2269 −1649 −2058 −2099 −2410 −719 3135 −1948 −3282−3046 91 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 86(V) −1750 −1296 −4319 −3957 −1765 −4038 −3733 2364 −3826 −619 −543−3716 −3869 −3685 −3902 −3354 −1743 3012 −3265 −2817 92 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * *  87(S) 923 −962 −2348 −2422−3132 −1207 −2248 −2850 −2440 −3140 −2285 −1624 −1954 −2158 −2477 3171−758 −1896 −3362 −3103 93 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * *  88(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505−2621 −4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −355294 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  89(G)−2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320−3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 95 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * *  90(I) −1880 −1493 −4193 −3724−953 −3837 −2980 3251 −3420 257 2372 −3485 −3608 −3005 −3310 −3087 −1840617 −2373 −2155 96 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  91(S) 2150 −939 −2407 −2415 −3075 −1197 −2205 −2781 −2384−3065 −2205 −1613 −1936 −2105 −2436 2652 −729 −1850 −3306 −3049 97 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  92(M) −979−1455 −1242 −1122 −1434 −1860 −1131 −1171 −974 −1285 4091 2176 −2226−1017 −1187 −1166 −1086 −1063 −1929 −1345 98 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * *  93(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 99 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  94(T) −959 −1691 −1249 −949 −2563 −1747 −929 −2093 1282 −2263−1554 −995 −2115 −600 −354 −1037 3152 −1726 −2494 −2098 100 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  95(E) −572 −1860 −2082213 −2107 −1461 −191 −1808 199 −116 −983 −127 318 1199 −269 −475 −517−1448 −2078 −1441 101 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * *  96(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 102 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *  97(M) −2406−2296 −3638 −3594 −1525 −3105 −2824 −1047 −3121 −596 5043 −3293 −3425−3046 −2996 −2911 −2552 −1398 −2513 −2207 103 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * *  98(R) −2097 −2786 −2688 −1415−3622 −2625 −555 −2964 2585 −2627 −1957 −1318 −2577 −137 3015 −1979−1791 −2732 −2469 −2363 104 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * *  99(Y) −3615 −2706 −4169 −4413 2626 −4044 −396−2535 −3993 −1939 −1985 −2747 −3930 −2852 −3446 −3296 −3494 −2686 3474252 105 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *100(S) −897 −1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686 −3497 −2780−1973 −2360 −2483 −2703 3465 −1316 −2413 −3310 −3025 106 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 101(L) −2871 −2457 −4231−4103 −1033 −3803 −3165 −541 −3734 3130 −31 −3935 −3797 −3286 −3484−3713 −2869 −1136 −2394 −2220 107 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 102(V) −1381 −1065 −3714 −3252 −1453 −3300−2646 1872 −3023 −615 −373 −2949 −3287 −2816 −3039 −2506 1346 2750 −2489−2087 108 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *103(S) −897 −1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686 −3497 −2780−1973 −2360 −2483 −2703 3465 −1316 −2413 −3310 −3025 109 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 104(R) −2957 −3022 −3318−2735 −3796 −2998 −1968 −3912 −846 −3631 −3157 −2611 −3280 −1724 4056−3026 −2913 −3650 −3096 −3185 110 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 105(E) −1719 −3572 2596 2779 −3767 −1632 −993−3700 −1241 −3578 −2920 −234 −2167 −666 −2090 −1380 −1789 −3182 −3742−2756 111 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *106(V) −1746 −1296 −4308 −3946 −1757 −4020 −3712 2190 −3811 −614 −539−3702 −3858 −3667 −3884 −3336 −1740 3098 −3250 −2803 112 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 107(I) −2091 −1746 −3971−3840 −1676 −3532 −3289 3684 −3581 −659 −693 −3562 −3674 −3445 −3521−3194 −2146 449 −2877 −2493 113 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 108(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735−2840 −3028 −3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405−3320 114 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *109(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621 −4365 −3956−1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 115 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 110(S) −352 2942 −2955−2957 −2876 −1254 −2382 −2573 −2692 −2927 −2128 −1827 −2001 −2405 −26073103 −778 −1757 −3171 −2911 116 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 111(I) −2091 −1746 −3971 −3840 −1676 −3532 −32893684 −3581 −659 −693 −3562 −3674 −3445 −3521 −3194 −2146 449 −2877 −2493117 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 112(E)−2641 −3308 −896 3732 −3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531−2959 −1842 −2560 −2479 −2750 −3722 −3563 −3385 118 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 113(T) 1556 −936 −2493 −2457−2805 −1256 −2159 −2210 −2319 −2681 −1932 −1656 −1974 −2089 −2352 −5983235 −1547 −3111 −2847 119 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 114(C) 1784 2119 −2013 −1532 −1093 −1580 −1089 −436 −1322−937 −273 1093 −1932 −1127 −1472 −748 −515 1585 −1536 −1163 120 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 115(M) 1831 2019−2596 −2038 −605 −1979 −1126 244 −1727 −359 2501 −1655 −2145 −1435 −1683−1106 −557 1087 −1153 −804 121 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 116(Q) −987 −2211 −43 −62 −2833 2229 −691 −2616−407 −2604 −1797 1197 −1917 2260 −858 −880 −1045 −2139 −2772 −2099 122 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 117(G) 2313−1042 −2391 −2526 −3250 2601 −2372 −2972 −2637 −3257 −2407 −1721 −2032−2310 −2646 −662 −859 −2003 −3434 −3247 123 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 118(Q) −914 −2350 −48 1661 −2621 −15712504 −2400 68 −2331 −1486 −201 −1796 2646 −351 −754 −865 −1984 −2463−1787 124 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *119(W) −517 −1294 −733 −183 −1062 −1605 −234 −1037 19 −1207 −456 1435−1690 33 756 411 −454 −819 3340 1286 125 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 120(M) 410 −469 −2417 −1828 −341 −2041−897 195 −1513 −156 3130 −1534 −2102 −1230 −1484 −1117 −507 954 −8942253 126 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *121(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621 −4365 −3956−1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 127 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 122(G) 2142 −930 −2334−2298 −3100 2237 −2139 −2842 −2302 −3074 −2187 −1557 −1909 −2010 −23971136 −701 −1871 −3308 −3053 128 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 123(V) −1514 −1144 −3950 −3459 1821 −3487 −25772274 −3208 −209 −87 −3112 −3362 −2864 −3118 −2680 −1476 2426 −2194 −1786129 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 124(V)−1743 −1294 −4292 −3873 −1511 −3988 −3433 2287 −3712 598 −319 −3626−3774 −3456 −3716 −3260 −1717 2790 −2931 −2577 130 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 125(A) 2911 −954 −2808 −2665−2115 −1577 −2196 −575 −2445 −1646 −1202 −1906 −2208 −2218 −2451 −901−876 1294 −2727 −2394 131 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 126(I) −1764 −1323 −4298 −3936 −1668 −3994 −3655 3337−3783 −508 −462 −3689 −3838 −3608 −3835 −3311 −1759 1847 −3164 −2747 132— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 127(G) −1157−1705 −2169 −2375 −3654 3021 −2534 −3611 −2730 −3741 −2984 −2024 2418−2475 −2826 −1361 −1555 −2705 −3513 −3509 133 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 128(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 134 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 129(C) −2476 5735 −4102 −4358 −3712 −2763 −3545−3518 −4167 −3859 −3569 −3631 −3363 −4030 −3832 −2793 −2860 −3158 −3464−3718 135 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *130(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621 −4365 −3956−1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 136 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 131(K) −2620 −2961−2461 −2046 −3743 −2791 −1570 −3603 3784 −3387 −2839 −2048 −3039 −1260−465 −2604 −2536 −3331 −3001 −2988 137 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 132(N) −2171 −2655 −1458 −1748 −3334−2364 −2267 −3943 −2365 −3936 −3437 4205 −2932 −2205 −2608 −2224 −2439−3392 −3253 −2909 138 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 133(M) −2406 −2296 −3638 −3594 −1525 −3105 −2824 −1047 −3121−596 5043 −3293 −3425 −3046 −2996 −2911 −2552 −1398 −2513 −2207 139 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 134(P) −2931−2878 −3420 −3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165 −3491 4225−3781 −3695 −3182 −3279 −4087 −3594 −4064 140 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 135(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 141 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 136(A) 2180 −935 −2286 −2196 −3057 1098 −2058 −2796−2174 −3021 −2134 −1516 −1898 −1906 −2302 2146 −689 −1849 −3256 −2983142 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 137(M)−1799 −1433 −4142 −3579 −669 −3668 −2608 1558 −3293 1235 3799 −3296−3401 −2717 −3088 −2843 −1726 1156 −2002 −1868 143 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 138(I) −2091 −1746 −3971 −3840−1676 −3532 −3289 3684 −3581 −659 −693 −3562 −3674 −3445 −3521 −3194−2146 449 −2877 −2493 144 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 139(A) 3103 −1036 −2445 −2572 −3222 1051 −2380 −2930−2650 −3226 −2381 −1739 −2034 −2327 −2648 −664 −857 −1981 −3412 −3228145 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 140(M)−2325 −1891 −4598 −4012 −498 −4222 −3013 1242 −3722 1864 3929 −3855−3711 −2910 −3414 −3439 −2215 −299 −2076 −2098 146 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 141(A) 3103 −1036 −2445 −2572−3222 1051 −2380 −2930 −2650 −3226 −2381 −1739 −2034 −2327 −2648 −664−857 −1981 −3412 −3228 147 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 142(R) −1588 −2442 −1399 −953 −3069 −2171 −708 −2795 373−2625 −1916 1858 −2357 −324 3294 −1520 −1505 −2453 −2523 −2186 148 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 143(M) −1448−1256 −3396 −2819 −474 −3024 −1923 175 −2473 2225 2756 −2574 −2922 −2063−2375 −2153 952 −151 −1599 −1410 149 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 144(N) −1662 −3306 2055 78 −3621 −1643−1040 −3622 −1272 −3531 −2870 3477 −2182 −724 −2071 −1371 −1757 −3092−3633 −2700 150 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 145(I) −1066 −921 −2828 −2239 −1041 −2675 −1601 2235 −1668−455 −92 −2067 −2692 −1688 1701 −1795 −1024 1960 −1771 −1396 151 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 146(P) −2931 −2878−3420 −3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165 −3491 4225 −3781−3695 −3182 −3279 −4087 −3594 −4064 152 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 147(S) 1568 −940 −2267 −2192 −3082 1101−2068 −2826 −2185 −3049 −2159 −1515 −1901 −1915 −2313 2603 −694 −1866−3279 −3006 153 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 148(I) −1880 −1492 −4195 −3728 −963 −3841 −2991 3272 −3425 2462277 −3490 −3613 −3014 −3317 −3092 −1841 628 −2385 −2163 154 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 149(F) −2204 −1797−3724 −3473 3206 −3383 −628 −1077 −3092 −746 3167 −2502 −3309 −2372−2792 −2535 −2120 −1245 28 2460 155 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 150(V) 1265 −1028 −3200 −2994 −1833 −2150−2480 417 −2771 −1122 −818 −2349 −2640 −2559 −2766 −1464 −1118 3028−2700 −2325 156 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 151(Y) −3482 −2868 −3701 −3919 238 −3552 −1112 −3000 −3638−2516 −2526 −3027 −3772 −3101 −3341 −3418 −3527 −3071 −441 4711 157 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 152(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 158 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 153(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 159 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 154(T) −359 −976 −2225 −2229 −2900 −1242 −2074−2560 −2170 −2875 −2064 −1561 −1958 −1969 −2247 1110 3375 −1760 −3152−2850 160 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *155(I) −2091 −1746 −3971 −3840 −1676 −3532 −3289 3684 −3581 −659 −693−3562 −3674 −3445 −3521 −3194 −2146 449 −2877 −2493 161 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 156(H) 861 −1924 −384 1010−2260 −1477 1787 −1974 1769 −1918 −1022 −120 −1566 362 697 −417 −459−1557 −2073 −1446 162 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 157(P) −655 −1502 −711 −557 −2204 −1463 2143 −2122 −586 −2233−1445 −688 2941 −560 −941 855 −805 −1657 −2369 −1763 163 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 158(G) −2594 −2690 −3304−3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779−2839 −2981 −4004 −3668 −4222 164 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 159(H) −744 −2193 −114 1118 −2513 −1512 2486−2252 1178 −2183 −1308 2230 −1689 180 −233 −598 −687 −1823 −2335 −1670165 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 160(W)−2672 −2139 −3850 −3748 941 −3611 −469 −1691 −3306 1047 −1217 −2551−3534 −2514 −2960 −2788 −2577 −1799 4205 3466 166 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 161(K) 386 −1981 779 279 −2295−1403 −114 −2043 2059 −1991 −1082 941 −1536 1263 −211 −384 −457 −1602−2161 −1476 167 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 162(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 168 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 163(K) −1144−2365 −912 2048 −2856 −1912 −326 −2459 2267 −2295 −1482 −556 −1989 1081334 −1013 −1014 −2093 −2324 −1881 169 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 164(D) −1091 −2610 2941 174 −2957 −1527−595 −2750 1084 −2696 −1877 −176 −1885 −206 −1006 740 −1098 −2288 −2880−2105 170 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *165(L) −2387 −1922 −4674 −4155 −617 −4366 −3250 1889 −3865 2650 558−4023 −3847 −3098 −3586 −3647 −2296 −38 −2247 −2224 171 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 166(N) −1021 −2427 1806 133−2870 −1499 −635 −2647 −521 −2640 −1825 2171 −1874 −255 −1124 −860 2122−2184 −2853 −2090 172 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 167(I) −1830 −1390 −4327 −3873 −1210 −3994 −3274 2967 −36781259 −30 −3633 −3730 −3283 −3604 −3249 −1791 1570 −2661 −2417 173 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 168(V) −1771 −1603−3750 −3689 −2037 −3050 −3231 403 −3479 −1154 −1076 −3246 −3399 −3383−3437 −2628 −1917 3536 −3074 −2677 174 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 169(S) −897 −1462 −2333 −2543 −3185−1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360 −2483 −2703 3465 −1316−2413 −3310 −3025 175 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 170(A) 2440 −824 −2371 −2082 −1993 −1344 −1704 −1264 −1899−1832 −1137 −1517 −1946 −1674 −2005 1075 −641 1474 −2390 −2055 176 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 171(F) −3342−2776 −4026 −4232 4354 −3545 −1431 −2315 −4038 −1801 −1900 −3299 −3780−3350 −3645 −3490 −3420 −2566 −739 349 177 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 172(E) −2641 −3308 −896 3732 −3966 −2458−2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560 −2479 −2750 −3722−3563 −3385 178 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 173(A) 2966 −1031 −2429 −2551 −3222 1544 −2368 −2934 −2633−3225 −2377 −1727 −2028 −2309 −2637 −656 −850 −1980 −3412 −3224 179 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 174(V) −1769−1342 −4255 −3793 −1216 −3901 −3162 1633 −3589 1486 −51 −3537 −3667−3214 −3518 −3143 −1731 2692 −2609 −2345 180 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 175(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 181 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 176(Q) −729 −2116 −413 1096 −2484 −1587 1599 −2186 1695 −2094−1219 −223 −1698 2418 90 −599 −649 −1770 −2213 −1615 182 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 177(W) −1652 −1707 −2340−1879 1996 −2733 2013 −1398 1758 −1386 −938 −1641 −2751 −1364 −1762−1780 −1577 −1325 3577 2136 183 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 178(T) −421 −753 −1251 −704 −846 −1670 −535 894−548 −690 −1 1376 −1791 −421 −846 373 1461 858 −1236 −812 184 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 179(H) 1498 −1593−504 15 −1895 −1484 2279 −1559 1119 −1640 −810 −242 −1611 194 −171 −462815 −1231 −1914 −1340 185 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 180(G) −1515 −2130 −1298 −1450 −2658 3285 2212 −3276−1691 −3291 −2638 −1524 −2562 −1662 −1925 −1600 −1764 −2713 −2804 −2234186 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 181(K)−528 −2010 1346 1082 −2329 −1408 −118 −2080 1475 −2018 −1108 1161 −1543331 1052 −394 −471 −1632 −2181 −1494 187 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 182(M) −1894 −1521 −4170 −3679 −840−3793 −2866 2827 −3360 375 3445 −3437 −3555 −2902 −3223 −3028 −1846 470−2249 −2059 188 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 183(T) −670 −1758 1731 −141 −2591 −1399 −691 −2319 −499 −2384−1543 −387 −1786 −316 −1016 1576 2044 −1811 −2624 −1981 189 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 184(E) 345 −2074 9251994 −2378 −1408 −177 −2135 922 −2084 −1183 −38 641 264 −356 −444 −536−1690 −2261 −1556 190 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 185(E) −1493 −2900 93 3174 −2903 −1743 1987 −3042 −646 −2957−2238 −411 −2146 −506 −1121 −1272 −1503 −2629 −2905 −2134 191 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 186(D) −1293 −29592673 2121 −3219 −1546 −713 −3043 −707 −2974 −2191 −158 −1967 −342 −1394−1043 701 −2567 −3172 −2311 192 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 187(F) −1137 −905 −3250 −2707 2365 −2647 −1016 −34−2336 1239 267 −2150 −2626 −1861 −2133 −1752 −1069 1461 −599 1844 193 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 188(K) −479−1713 −409 1031 −1925 −1467 1755 −1650 1844 −349 −827 −140 −1556 319 −75−403 −411 −1301 −1900 843 194 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 189(G) 433 −2144 52 1047 −2717 2303 −615 −2467 −442−2482 −1655 1123 −1828 −233 −995 −763 −923 −2000 −2710 −2005 195 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 190(V) −1752 −1320−4254 −3806 −1311 −3916 −3232 1701 −3614 1188 −140 −3551 −3693 −3280−3568 −3166 −1718 2833 −2703 −2409 196 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 191(E) −1199 −1750 −734 2668 −1820 −2038−1068 1892 −867 −1273 −897 −922 −2295 −797 −1238 −1340 −1197 −426 −2325−1789 197 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *192(C) −1182 3528 −1398 −620 −2541 −2038 −358 −2093 1181 −2037 −1272−747 −2070 1553 2213 −1123 −1038 −1817 −2142 −1774 198 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 193(N) −1478 −2527 −261 −403−2011 −1837 2032 −2925 −735 −2845 −2195 3635 −2259 −721 −1085 −1352−1546 −2522 −2307 −1431 199 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 194(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735−2840 −3028 −3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405−3320 200 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *195(C) −1220 4911 −3609 −3314 −1440 −2525 −2482 1565 −2922 −706 −544−2678 −2896 −2710 −2836 −1869 −1375 379 −2371 −1957 201 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 196(P) −2931 −2878 −3420−3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165 −3491 4225 −3781 −3695−3182 −3279 −4087 −3594 −4064 202 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 197(G) −477 −1115 −1983 −2189 −3315 3154 −2272−3172 −2506 −3387 −2522 −1599 −2042 −2177 −2583 1217 −905 −2130 −3477−3225 203 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *198(A) 1653 −1347 −705 −249 −1969 −1385 −477 −1629 −159 −1759 −935 −4341285 1404 −586 −450 1019 −1243 −2070 −1522 204 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 199(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 205 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 200(S) 1870 −938 −2270 −2183 −3068 1488 −2056 −2810−2168 −3032 −2144 −1511 −1898 −1901 −2300 2236 −690 −1857 −3265 −2990206 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 201(C)−2476 5735 −4102 −4358 −3712 −2763 −3545 −3518 −4167 −3859 −3569 −3631−3363 −4030 −3832 −2793 −2860 −3158 −3464 −3718 207 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 202(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 208 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 203(G) −2594 −2690 −3304 −3623 −4328 3747 −3462−4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668−4222 209 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *204(M) −2406 −2296 −3638 −3594 −1525 −3105 −2824 −1047 −3121 −596 5043−3293 −3425 −3046 −2996 −2911 −2552 −1398 −2513 −2207 210 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 205(Y) −3590 −2700−4146 −4379 2092 −4028 −404 −2517 −3963 −1928 −1973 −2744 −3921 −2845−3431 −3284 −3474 −2669 336 4423 211 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 206(T) −1213 −1674 −2755 −2906 −3163−1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −1463 3819−2197 −3286 −3156 212 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 207(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028−3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405 −3320 213 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 208(N) −2171−2655 −1458 −1748 −3334 −2364 −2267 −3943 −2365 −3936 −3437 4205 −2932−2205 −2608 −2224 −2439 −3392 −3253 −2909 214 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 209(T) −1213 −1674 −2755 −2906−3163 −1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −14633819 −2197 −3286 −3156 215 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 210(M) −2355 −1988 −4343 −3834 −504 −4051 −2868 105 −33851451 4460 −3680 −3671 −2806 −3171 −3327 −2274 −474 −2039 −1925 216 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 211(S) 2150−939 −2407 −2415 −3075 −1197 −2205 −2781 −2384 −3065 −2205 −1613 −1936−2105 −2436 2652 −729 −1850 −3306 −3049 217 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 212(S) −344 −979 −2190 −2162 −2959 −1227−2042 −2651 −2116 −2934 −2100 −1526 −1941 −1909 −2222 2940 1775 −1804−3187 −2882 218 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 213(A) 3048 −932 −2480 −2533 −3075 −1200 −2274 −2765 −2501−3071 −2221 −1658 −1948 −2205 −2512 1225 −739 −1842 −3322 −3078 219 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 214(I) −1924−1546 −4067 −3658 2312 −3663 −2081 3030 −3367 150 99 −3197 −3492 −2821−3179 −2894 −1877 293 −1445 −692 220 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 215(E) −2641 −3308 −896 3732 −3966 −2458−2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560 −2479 −2750 −3722−3563 −3385 221 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 216(A) 2389 −814 −2506 −2162 −1696 −1545 −1698 −499 −1942−1398 −813 −1640 −2076 −1723 −2027 −806 1148 1559 −2200 −1856 222 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 217(M) −2576 −2118−4725 −4165 −461 −4430 −3165 99 −3811 2513 3454 −4075 −3839 −2978 −3488−3704 −2457 −591 −2111 −2145 223 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 218(G) −2594 −2690 −3304 −3623 −4328 3747−3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004−3668 −4222 224 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 219(M) −2313 −1968 −4258 −3765 −518 −3966 −2806 98 −3289 12924523 −3599 −3636 −2769 −3097 −3249 −2243 −457 −2026 −1874 225 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 220(S) −897 −1462−2333 −2543 −3185 −1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360 −2483−2703 3465 −1316 −2413 −3310 −3025 226 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 221(L) −2631 −2159 −4786 −4228 −462−4506 −3231 96 −3878 2828 2482 −4157 −3880 −3016 −3541 −3793 −2509 −608−2134 −2182 227 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 222(P) −1501 −1778 −2473 −2371 −1710 −2311 −2045 −1321 −2060827 −1068 −2173 3594 −2082 −2130 −1799 −1699 −1373 −2373 −1942 228 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 223(Y) −1068−1670 −865 −836 −631 1198 −767 −1828 −1059 −1914 −1304 692 −2203 −906−1387 −1136 −1163 −1566 −1185 3670 229 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 224(S) −897 −1462 −2333 −2543 −3185−1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360 −2483 −2703 3465 −1316−2413 −3310 −3025 230 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 225(S) 1172 −954 −2367 −2422 −3120 −1204 −2237 −2835 −2426−3122 −2265 −1621 −1948 −2145 −2467 3107 −749 −1884 −3349 −3092 231 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 226(S) −342−975 −2176 −2124 −2912 −1229 −2003 −2594 −2067 −2878 −2048 −1510 −1936−1866 −2184 2553 2492 −1773 −3143 −2833 232 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 227(M) −720 −1440 −710 −343 −1228 −16932436 −1209 −132 −1364 3099 1904 −1852 −183 −458 −776 −680 −1004 −1540−890 233 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *228(P) 2240 −1100 −2241 −2293 −3037 −1346 −2188 −2683 −2317 −2986 −2210−1663 3041 −2093 −2391 −722 −895 −1893 −3243 −2998 234 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 229(A) 2958 −1235 −1299−1377 −2868 −1345 −1673 −2580 −1661 −2843 −2054 1555 −1995 −1468 −1921−715 −888 −1871 −3064 −2630 235 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 230(E) −509 −1046 −884 1564 −1116 −1669 −441 −485−283 250 −206 −577 689 −200 −656 −670 −459 1290 −1467 −995 236 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 231(D) −1203 −24122595 −117 −3286 −1536 −1057 −3176 −1165 −3186 −2436 −428 −2068 −736−1824 2377 −1366 −2578 −3334 −2552 237 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 232(Q) 954 −1983 −100 971 −2337 177 −267−2067 81 −2060 −1189 −125 −1637 2600 −418 −514 −597 −1649 −2268 −1597238 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 233(E)−2641 −3308 −896 3732 −3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531−2959 −1842 −2560 −2479 −2750 −3722 −3563 −3385 239 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 234(K) −2620 −2961 −2461 −2046−3743 −2791 −1570 −3603 3784 −3387 −2839 −2048 −3039 −1260 −465 −2604−2536 −3331 −3001 −2988 240 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 235(R) 377 −1802 −415 988 −2095 −1474 −95 −17861452 −1785 −911 −135 −1560 343 1555 −409 −431 376 −1986 −1375 241 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 236(D) 1083 −15652662 −244 −1941 −1573 −679 612 −527 −1651 −980 −490 −1869 −358 −1003−771 −766 −903 −2208 −1633 242 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 237(E) −1225 −2868 1894 1948 −3149 −1532 −671 −2975−630 −2902 −2101 −150 −1935 −293 −1299 1884 −1241 −2496 −3093 −2248 243— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 238(C) 13753262 −2620 −2108 −827 −1866 −1267 1631 −1811 −599 −10 −1674 −2137 −1531−1786 −1034 790 249 −1361 −1010 244 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 239(E) 635 −1796 1055 1761 −2018 −1464 −2631191 28 −1767 −946 −148 −1637 135 −481 −520 −553 −1300 −2077 −1441 245 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 240(E) 593−2044 −252 2548 −2437 −1542 −329 −2133 151 −2120 −1274 −244 −1738 89 946−646 −717 −1734 −2305 −1686 246 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 241(S) 1884 −835 −1962 −1576 −1634 −1436 −1320 1041−1409 −1453 −781 −1293 −1922 −1241 −1606 1973 −597 −669 −2036 −1656 247— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 242(G) 2267−1043 −2388 −2526 −3253 2642 −2373 −2975 −2639 −3260 −2410 −1722 −2033−2311 −2648 −663 −860 −2005 −3436 −3250 248 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 243(R) −876 −2087 −829 1490 −2474 −1766−229 −2106 1269 −44 −1198 −424 −1829 205 2225 −775 −768 −1753 −2143−1647 249 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *244(V) 2339 −967 −2970 −2766 −1878 −1847 −2252 32 −2541 −1299 −918 −2087−2399 −2316 −2545 −1157 −971 2345 −2605 −2251 250 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 245(I) −1827 −1398 −4307 −3831−1099 −3939 −3142 2286 −3619 1835 69 −3579 −3671 −3177 −3511 −3178 −17811918 −2524 −2310 251 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 246(V) −1178 −1448 −1943 −1452 −1776 −2261 −1140 −227 1866−1260 −816 −1444 −2448 −902 −540 −1496 −1176 2697 −2161 −1764 252 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 247(E) −508 −1976840 1547 −2280 −1393 −117 −2029 1400 −1984 −1077 1158 −1531 330 −253−378 −454 262 −2163 −1471 253 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 248(M) 1703 −991 −2901 −2342 −528 −2567 −1550 166−2031 1544 2668 −2104 −2591 −1715 −2010 −1685 −1052 −12 −1442 −1177 254— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 249(I) −1947−1516 −4385 −3885 −916 −4013 −3118 2193 −3656 2186 257 −3656 −3687 −3109−3494 −3250 −1889 1383 −2397 −2258 255 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 250(E) −1322 −2647 −272 2491 −3071 −1811−576 −2759 2306 −2633 −1854 −464 −2066 −175 −177 −1144 −1256 −2368 −2692−2140 256 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *251(K) −1395 −2059 −1711 −1014 −2215 −2218 −641 −1709 3021 −1652 2578−1075 −2303 −282 287 −1423 −1283 −1603 −2159 −1803 257 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 252(D) −1285 −2888 2677 176−3210 1189 −737 −3047 −715 −2977 −2195 −190 −1979 2106 −1379 −1050 −1315−2564 −3161 −2320 258 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 253(I) −2073 −1632 −4434 −3975 −911 −4130 −3238 3164 −37061451 244 −3779 −3785 −3187 −3557 −3413 −2021 546 −2449 −2273 259 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 254(K) −1570 −2144−1887 −1191 −2098 −2363 −750 −1603 3034 938 −1112 −1231 −2436 −408 215−1616 −1443 −1580 −2166 −1804 260 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 255(P) −2931 −2878 −3420 −3706 −4181 −2925−3468 −4621 −3859 −4490 −4165 −3491 4225 −3781 −3695 −3182 −3279 −4087−3594 −4064 261 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 256(R) −928 −1705 −1507 −1055 −2761 −1730 −896 −2490 −44 −2489−1723 −1042 −2102 −543 2614 2258 −1053 −1998 −2546 −2158 262 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 257(D) −1280 −2865 3154175 −3194 −1547 −743 −3034 −728 −2971 −2194 −190 −1979 1342 −1391 553−1316 −2552 −3161 −2317 263 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 258(I) −1997 −1562 −4355 −3927 −1042 −4066 −32613343 −3654 937 97 −3718 −3783 −3239 −3555 −3364 −1959 702 −2549 −2295264 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 259(M)−2252 −1821 −4572 −3991 −530 −4164 −2990 2068 −3709 1993 3197 −3808−3685 −2916 −3406 −3378 −2149 −172 −2084 −2091 265 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 260(T) −1213 −1674 −2755 −2906−3163 −1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −14633819 −2197 −3286 −3156 266 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 261(R) −2131 −2786 −2704 −1460 −3618 −2638 −587 −29761735 −2645 −1985 −1353 −2603 −173 3492 −2020 −1828 −2748 −2484 −2384 267— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 262(K) −1349−2635 −381 2083 −3083 −1857 −565 −2750 2690 −2612 −1837 −514 −2090 −161−61 −1178 −1271 −2369 −2655 −2138 268 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 263(A) 2821 −932 −2451 −2472 −3065 −1198−2233 −2763 −2434 −3056 −2201 −1633 −1940 −2147 −2468 1831 −730 −1840−3305 −3055 269 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 264(F) −2063 −1686 −4037 −3677 3437 −3644 −1706 2063 −3359 13567 −3095 −3486 −2739 −3127 −2876 −2012 −83 −1038 −158 270 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 265(E) −2641 −3308 −8963732 −3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560−2479 −2750 −3722 −3563 −3385 271 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 266(N) −1662 −3306 2055 78 −3621 −1643 −1040−3622 −1272 −3531 −2870 3477 −2182 −724 −2071 −1371 −1757 −3092 −3633−2700 272 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *267(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028 −3257 −2662−2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405 −3320 273 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 268(I) −1760 −1307−4325 −3962 −1735 −4042 −3726 3135 −3828 −579 −515 −3722 −3869 −3673−3896 −3359 −1752 2276 −3240 −2806 274 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 269(T) 1428 −904 −2334 −2158 −2747 −1206−1940 −2392 −2037 −2678 −1846 −1504 −1896 −1809 −2163 902 3001 −1635−2999 −2705 275 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 270(V) −1745 −1300 −4286 −3858 −1446 −3967 −3370 2358 −3688852 −261 −3606 −3749 −3403 −3673 −3232 −1717 2643 −2856 −2524 276 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 271(V) −1404 −1072−3766 −3305 −1464 −3356 −2696 2276 −3080 −616 −379 −3001 −3325 −2870−3091 −2563 1344 2521 −2516 −2113 277 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 272(M) 866 −1113 −2656 −2412 −1322 −1920−1883 −487 −2061 −587 4451 −1950 −2387 −1928 −2078 −1220 −1053 −498−2134 −1803 278 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 273(A) 2601 −957 −2898 −2711 −1943 −1740 −2211 −165 −2487−1406 −1001 −2008 −2320 −2260 −2494 −1053 −929 1990 −2626 −2279 279 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 274(L) −1171−983 −3266 −2733 −796 −2795 −1888 590 −2418 2001 198 −2418 −2816 −2106−2362 −1944 965 1777 −1724 −1426 280 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 275(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 281 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 276(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 282 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 277(S) −897−1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360−2483 −2703 3465 −1316 −2413 −3310 −3025 283 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 278(T) −1213 −1674 −2755 −2906 −3163−1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −1463 3819−2197 −3286 −3156 284 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 279(N) −2171 −2655 −1458 −1748 −3334 −2364 −2267 −3943 −2365−3936 −3437 4205 −2932 −2205 −2608 −2224 −2439 −3392 −3253 −2909 285 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 280(A) 3134−934 −2491 −2567 −3083 −1203 −2300 −2766 −2540 −3082 −2237 −1672 −1954−2240 −2537 874 −747 −1844 −3333 −3093 286 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 281(V) −984 −1045 −3169 −2909 −1709−2304 −2404 531 −2643 −988 −697 −2378 −2722 −2480 −2661 −1601 1504 3014−2588 −2201 287 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 282(L) −2631 −2159 −4786 −4228 −462 −4506 −3231 96 −3878 28282482 −4157 −3880 −3016 −3541 −3793 −2509 −608 −2134 −2182 288 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 283(H) −3205 −3079−2723 −2890 −2110 −3046 5295 −4135 −2617 −3813 −3561 −2886 −3482 −2833−2620 −3291 −3356 −3895 −2397 −1681 289 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 284(L) −1623 −1338 −3726 −3164 −251−3255 −1820 1373 −2808 2371 514 −2785 −3086 −2281 −2613 −2389 −1543 −161−1311 1782 290 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 285(L) −2333 −1873 −4640 −4127 −650 −4326 −3241 2176 −38432519 523 −3982 −3833 −3105 −3579 −3604 −2247 56 −2268 −2230 291 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 286(A) 3438 −1472−2846 −3040 −3287 −1726 −2735 −2840 −3028 −3257 −2662 −2236 −2447 −2798−2944 −1216 −1387 −2183 −3405 −3320 292 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 287(M) −1886 −1507 −4178 −3693 −877−3806 −2901 3008 −3380 335 3109 −3451 −3570 −2934 −3251 −3044 −1840 524−2288 −2089 293 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 288(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028−3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405 −3320 294 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 289(H) −1490−2484 −362 −476 −1816 −1880 4320 −2854 −684 −2770 −2133 2185 −2285 −728−1000 −1377 −1550 −2475 −2146 −1255 295 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 290(A) 2439 −911 −2326 −2131 −2811 −1197−1934 −2480 −2011 −2745 −1898 −1490 −1888 −1785 −2153 1898 1073 −1682−3044 −2749 296 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 291(I) 2038 −985 −3388 −2919 −1320 −2893 −2277 2155 −2677 −587−297 −2593 −2992 −2450 −2697 −2087 −1208 1681 −2229 −1846 297 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 292(G) −1243 −2769311 1902 −3172 1980 −744 −2992 −697 −2936 −2152 1923 −1974 −377 −1331−1030 −1284 −2506 −3125 −2308 298 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 293(V) −1738 −1298 −4281 −3921 −1737 −3979−3665 1917 −3774 −601 −528 −3671 −3834 −3628 −3843 −3293 −1735 3205−3215 −2770 299 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 294(E) −833 −2344 1092 2412 −2643 −1464 −386 −2413 −146 −2369−1505 −96 562 29 −717 −666 862 −1966 −2562 −1818 300 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 295(W) −1380 −1116 −3614 −30261322 −2981 −1582 1966 −2661 1775 556 −2562 −2865 −2117 −2424 −2098 −1302−187 2908 −629 301 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 296(T) −350 −973 −2204 −2178 −2893 −1236 −2035 −2561 −2117−2862 −2043 −1536 −1946 −1916 −2214 1618 3198 −1758 −3137 −2831 302 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 297(L) −1443−1269 −3144 −2576 −528 −3014 −1816 1945 −2155 2102 508 −2422 −2899 1193−2133 −2129 −1369 −50 −1616 −1384 303 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 298(D) −1826 −3682 3559 1199 −3883 −1662−1073 −3846 −1391 −3720 −3110 −272 −2222 −760 −2283 −1471 −1913 −3321−3864 −2864 304 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 299(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621−4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 305 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 300(F) −3342−2776 −4026 −4232 4354 −3545 −1431 −2315 −4038 −1801 −1900 −3299 −3780−3350 −3645 −3490 −3420 −2566 −739 349 306 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 301(Q) −1048 −2608 205 2170 −2893 −1535−505 −2680 −255 −2604 −1769 1814 −1849 2272 −789 −848 −1028 −2228 −2770−2013 307 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *302(R) 1083 −1687 691 135 −2058 −1406 −178 −1755 214 −1793 −924 −145−1553 247 1670 −383 1217 −1367 −2031 −1404 308 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 303(I) −1915 −1536 −4077 −36672027 −3678 −2155 3137 −3381 144 94 −3225 −3506 −2848 −3202 −2914 −1871345 −1522 −791 309 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 304(R) −689 −2015 −494 24 −2395 −1582 −184 −2087 444 −2020−1151 1161 −1687 1832 2131 626 −614 −1684 −2156 −1573 310 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 305(D) 387 −1967 16001359 −2275 −1391 1561 −2025 282 −1976 −1067 −25 −1525 342 1024 −369 −443−1584 −2152 −1462 311 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 306(R) −1460 −2315 −1793 −887 −2832 −2237 −431 −2288 2193−2199 −1473 −946 −2245 −20 2706 −1394 −1275 591 −2248 −1961 312 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 307(V) −941 −1027−3099 −2832 −1692 −2234 −2324 470 −2565 −1003 −695 −2305 −2663 −2399−2587 −1527 1858 2876 −2536 −2152 313 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 308(P) −2931 −2878 −3420 −3706 −4181−2925 −3468 −4621 −3859 −4490 −4165 −3491 4225 −3781 −3695 −3182 −3279−4087 −3594 −4064 314 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 309(V) −1090 −1215 −2097 −1824 −819 −2221 2699 −287 −1392−1027 −591 −1674 −2482 −1446 −1482 −1482 −1143 2879 −1420 −707 315 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 310(L) −2439−1972 −4702 −4181 −588 −4401 −3258 1582 −3881 2757 587 −4061 −3862 −3093−3590 −3689 −2344 −130 −2230 −2217 316 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 311(C) 2157 4166 −3012 −2973 −2780 1022−2337 −2398 −2724 −2744 −1930 −1786 −1943 −2372 −2623 −540 −692 −1624−3091 −2881 317 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 312(D) −1732 −3453 3468 99 −3733 −1645 −1066 −3747 −1356 −3641−3008 1690 −2201 −755 −2209 −1416 −1833 −3208 −3752 −2776 318 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 313(L) −2477 −2023−4713 −4122 1592 −4329 −2920 72 −3835 2593 2472 −3948 −3754 −2914 −3466−3550 −2350 −634 −1927 −1830 319 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 314(K) −2620 −2961 −2461 −2046 −3743 −2791−1570 −3603 3784 −3387 −2839 −2048 −3039 −1260 −465 −2604 −2536 −3331−3001 −2988 320 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 315(P) −2931 −2878 −3420 −3706 −4181 −2925 −3468 −4621 −3859−4490 −4165 −3491 4225 −3781 −3695 −3182 −3279 −4087 −3594 −4064 321 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 316(S) −897−1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360−2483 −2703 3465 −1316 −2413 −3310 −3025 322 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 317(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 323 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 318(K) 2 −2257 −1073 −374 −2740 −1908 −278 −2339 2328 −2192−1373 −562 −1953 2273 1344 −952 −933 −1980 −2234 −1799 324 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 319(Y) −3482 −2868−3701 −3919 238 −3552 −1112 −3000 −3638 −2516 −2526 −3027 −3772 −3101−3341 −3418 −3527 −3071 −441 4711 325 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 320(M) −1559 −1267 −3829 −3380 −1103−3357 −2655 805 −3067 −64 3046 −3065 −3326 −2779 −3011 −2591 −1556 2855−2312 −1998 326 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 321(M) 1225 −469 −2256 −1679 1656 −1926 −870 90 −1396 −2102763 −1424 −2028 −1129 −1411 −1008 712 154 −951 −586 327 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 322(T) −738 −2094 −84 1704−2416 −1495 −317 −2135 61 −2127 −1275 −163 −1704 1857 −405 −613 1930−1734 −2331 −1668 328 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 323(D) −1746 −3458 3540 90 −3744 −1650 −1081 −3767 −1381 −3662−3036 1386 −2211 −772 −2239 −1429 −1850 −3226 −3765 −2789 329 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 324(L) −2451 −1983−4707 −4186 −582 −4409 −3259 1510 −3884 2778 592 −4069 −3865 −3091 −3590−3698 −2355 −150 −2226 −2214 330 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 325(H) −2923 −2573 −2959 −2926 826 −3449 4553−2508 −2463 −2054 −1948 −2279 −3499 −2191 −2397 −2761 −2855 −2540 1232920 331 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *326(K) 373 −1957 −342 1025 −2297 −1472 −98 −2018 2111 −1954 −1056 906−1570 352 685 −424 −473 −1592 −2105 −1469 332 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 327(V) 1739 −1008 −3509 −3043−1376 −3028 −2406 1765 −2807 −615 −334 −2718 −3093 −2585 −2823 −2226−1263 2376 −2322 −1931 333 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 328(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761−3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222334 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 329(G)−2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320−3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 335 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 330(I) −1758 −1302 −4331 −3970−1756 −4054 −3748 2976 −3840 −603 −533 −3731 −3877 −3693 −3914 −3372−1750 2505 −3265 −2824 336 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 331(P) −2931 −2878 −3420 −3706 −4181 −2925 −3468 −4621−3859 −4490 −4165 −3491 4225 −3781 −3695 −3182 −3279 −4087 −3594 −4064337 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 332(Q)1795 −1440 −730 −492 −2453 682 −812 −2151 −508 −2256 −1426 −624 −17962666 −901 −590 −689 −1636 −2510 −1971 338 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 333(V) −1771 −1603 −3750 −3689 −2037−3050 −3231 403 −3479 −1154 −1076 −3246 −3399 −3383 −3437 −2628 −19173536 −3074 −2677 339 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 334(M) −2355 −1988 −4343 −3834 −504 −4051 −2868 105 −3385 14514460 −3680 −3671 −2806 −3171 −3327 −2274 −474 −2039 −1925 340 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 335(K) −2620 −2961−2461 −2046 −3743 −2791 −1570 −3603 3784 −3387 −2839 −2048 −3039 −1260−465 −2604 −2536 −3331 −3001 −2988 341 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 336(Y) −1187 −974 −3186 −2638 −117 −2732−1255 1905 −2270 73 1977 −2217 −2699 −1882 −2144 −1841 −1124 71 −9073254 342 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *337(L) −2871 −2457 −4231 −4103 −1033 −3803 −3165 −541 −3734 3130 −31−3935 −3797 −3286 −3484 −3713 −2869 −1136 −2394 −2220 343 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 338(L) −2871 −2457−4231 −4103 −1033 −3803 −3165 −541 −3734 3130 −31 −3935 −3797 −3286−3484 −3713 −2869 −1136 −2394 −2220 344 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 339(K) −864 −1785 −860 −366 −2128 −1763−407 −1612 2624 −1800 −1045 629 −1900 −28 62 −851 −805 1127 −2064 −1581345 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 340(N)602 −1686 −275 1008 −1926 −1415 1528 −1618 244 −1673 −815 1897 −1530 299−244 −371 −391 322 −1934 −1306 346 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 341(G) −1709 −2639 1362 −690 −3785 3257 −1671−3805 −1946 −3792 −3137 −980 −2480 −1424 −2576 −1630 −1936 −3150 −3628−3155 347 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *342(F) −942 −799 −2828 −2226 1797 −2476 −1269 1109 581 1793 516 −1952−2453 −1557 −1815 −1558 −875 52 −1138 −794 348 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 343(L) −2451 −1983 −4707 −4186−582 −4409 −3259 1510 −3884 2778 592 −4069 −3865 −3091 −3590 −3698 −2355−150 −2226 −2214 349 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 344(H) −3205 −3079 −2723 −2890 −2110 −3046 5295 −4135 −2617−3813 −3561 −2886 −3482 −2833 −2620 −3291 −3356 −3895 −2397 −1681 350 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 345(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 351 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 346(D) −2784 −3432 4016 −1200−4140 −2466 −2197 −4505 −2621 −4365 −3956 −1551 −3014 −2039 −3232 −2593−2938 −4046 −3710 −3552 352 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 347(C) 774 4452 −2162 −1688 −1962 −1478 −1302 −1474−944 −1796 −1088 −1351 −1979 −1147 1684 −732 −719 −1116 −2225 −1881 353— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 348(L) −2387−1922 −4674 −4155 −617 −4366 −3250 1889 −3865 2650 558 −4023 −3847 −3098−3586 −3647 −2296 −38 −2247 −2224 354 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 349(T) −1213 −1674 −2755 −2906 −3163−1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −1463 3819−2197 −3286 −3156 355 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 350(C) −1489 2972 −4007 −3563 −1524 −3541 −2939 2612 −3350−617 −413 −3224 −3470 −3129 −3335 −2770 −1475 2269 −2657 −2248 356 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 351(T) −364−979 −2232 −2250 −2904 −1245 −2090 −2559 −2191 −2881 −2075 −1571 −1964−1991 −2260 905 3428 −1762 −3159 −2858 357 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 352(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 358 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 353(K) −1716 −2632 −2004 −1008 −3336 −2379 −444 −2764 2775−2484 −1756 −1035 −2357 2151 1811 −1592 −1477 −2481 −2391 −2172 359 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 354(T) −1213−1674 −2755 −2906 −3163 −1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600−2708 −2753 −1463 3819 −2197 −3286 −3156 360 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 355(V) −1771 −1339 −4275 −3816 −1235−3919 −3194 2139 −3617 1520 −66 −3558 −3681 −3244 −3547 −3164 −1733 2390−2634 −2369 361 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 356(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028−3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405 −3320 362 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 357(E) −2641−3308 −896 3732 −3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531 −2959−1842 −2560 −2479 −2750 −3722 −3563 −3385 363 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 358(N) −823 −1917 −96 1188 −2187−1547 −506 −1711 −265 −1955 −1191 2711 −1815 −144 −747 −757 −815 1140−2297 −1666 364 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 359(L) −2153 −1779 −4360 −3884 −675 −3965 −3012 392 −3561 2726467 −3673 −3662 −2955 −3355 −3239 −2102 1281 −2207 −2099 365 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 360(E) 1136 −2084 −1752027 −2436 −1510 −274 −2147 1525 −2118 −1254 −175 −1692 152 −251 −593−670 −1736 −2296 −1650 366 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 361(H) 893 −1761 1357 214 −2092 −1387 1862 −1810 229−1825 −942 −83 −1527 293 −273 640 793 −1409 −2050 −1397 367 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 362(I) 608 −458 −2776−2176 1666 −2202 −1113 1712 −1836 −222 338 −1782 −2245 −1512 −1731 −1292867 1366 −1036 −684 368 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 363(P) −922 −1912 1681 −141 −2123 −1604 −687 −1787 −550187 −1245 −427 2677 −363 −1049 −882 −947 −1524 −2338 −1711 369 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 364(D) −1692 −36053364 1256 −3770 −1599 −957 −3700 −1216 −3569 −2909 1025 −2138 −628 −2083−1346 −1761 −3174 −3765 −2738 370 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 365(Q) −877 −1646 −633 499 −1610 −1781 −505−1210 −63 1648 −649 −558 −1931 2241 −360 −907 −814 −1097 −1882 −1385 371— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 366(P) −648−2019 1139 203 −2354 −1436 −285 −2089 29 −2086 −1217 −114 1965 1445 −492−529 1244 −1672 −2300 −1616 372 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −571 −7108 −1646 −894−1115 −701 −1378 * * 367(R) −422 −1009 −851 −304 1406 −1496 −183 −740147 −894 −230 −440 775 21 2009 −539 −381 −568 −1136 −521 373 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −23 −6560 −7602 −894 −1115 −341 −2249 * * 368(D) 1472 −1668 1835−70 −2356 −1385 −511 −2062 −246 −2128 −1275 −318 1353 −118 −746 −526 425−1602 −2380 −1752 374 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 369(G) −1044 −2230 2141 −100 −3222 2291 −982 −3045 −1033 −3050−2258 −395 −1985 −644 −1669 858 −1207 −2428 −3250 −2493 375 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 370(Q) −2562 −2904−1886 −1971 −3251 −2661 −2079 −3690 −1565 −3469 −3081 −2107 −3091 4371−1665 −2585 −2674 −3411 −3077 −2821 376 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 371(D) −1275 −2955 2862 1330 −3205 −1556−670 −3029 1509 −2936 −2141 −158 −1955 −290 −1213 −1025 −1281 −2554−3111 −2272 377 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 372(V) −1738 −1298 −4281 −3921 −1737 −3979 −3665 1917 −3774−601 −528 −3671 −3834 −3628 −3843 −3293 −1735 3205 −3215 −2770 378 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 373(I) −2091−1746 −3971 −3840 −1676 −3532 −3289 3684 −3581 −659 −693 −3562 −3674−3445 −3521 −3194 −2146 449 −2877 −2493 379 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 374(M) −584 −1354 −847 −246 −1467 −16592505 −1087 212 −374 2571 −449 −1729 1171 1074 −634 −507 −876 −1617 −1128380 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 375(P)−910 −2031 −73 1195 −2792 −1488 −794 −2539 −629 −2588 −1788 −401 3005−439 −1131 612 −1014 −2050 −2815 −2151 381 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 376(W) −1588 −1300 −3783 −3197 −329−3245 −1926 2071 −2827 1901 558 −2822 −3072 −2297 −2616 −2381 −1508 −1113483 −1042 382 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 377(E) −1024 −2640 1844 2310 −2908 −1498 −505 −2711 −344 −2636−1791 −107 −1824 1521 −957 207 −1011 −2243 −2817 −2021 383 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 378(N) −826 −2349 1089227 −2651 −1487 −341 −2416 1494 −2346 −1475 2601 −1724 1005 −522 −657−787 −1968 −2511 −1791 384 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 379(P) 1932 −1116 −2232 −2301 −3058 −1358 −2206 −2706−2336 −3009 −2238 −1674 3274 −2114 −2406 −739 −914 −1913 −3260 −3019 385— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 380(V) −914−773 −2713 −2129 −712 −2505 −1388 1452 1084 1324 204 −1926 −2507 −1580−1808 −1591 −859 1713 −1424 −1081 386 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 381(Y) −1484 −2331 −1762 −887 −2436−2254 −420 −2325 2137 −2195 −1475 −949 −2258 −39 1983 −1411 −1295 −2075−2087 2868 387 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 382(E) 1256 −1890 −206 1353 −2196 −1401 −89 −1930 812 −1898−996 −45 547 1252 −162 −356 −414 −1507 −2083 −1416 388 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 383(Q) −752 −2272 1586 1407−2561 −1448 −308 −2329 −23 −2276 −1396 −71 −1677 1749 −577 −590 1569−1881 −2459 −1727 389 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 384(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 390 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 385(H) −964−2089 −200 −136 −2264 −1600 3833 −2320 −296 −2338 −1558 1362 1479 −276−699 −881 −992 −1924 −2364 −1652 391 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 386(L) −2451 −1983 −4707 −4186 −582−4409 −3259 1510 −3884 2778 592 −4069 −3865 −3091 −3590 −3698 −2355 −150−2226 −2214 392 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 387(Q) 1643 −1017 −1196 −721 −1189 −1714 −668 1336 −497 −907−297 −823 −1893 2044 −794 −784 −569 −339 −1579 −1135 393 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 388(I) −1760 −1308 −4323−3961 −1730 −4039 −3721 3156 −3825 −575 −512 −3720 −3867 −3669 −3893−3356 −1753 2241 −3236 −2802 394 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 389(L) −2871 −2457 −4231 −4103 −1033 −3803−3165 −541 −3734 3130 −31 −3935 −3797 −3286 −3484 −3713 −2869 −1136−2394 −2220 395 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 390(K) −1259 −2115 −1267 −676 −970 −2105 1794 −2040 2549 −1955−1282 −808 −2165 −167 114 −1192 −1140 −1801 −1301 2517 396 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 391(G) −2594 −2690−3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748−3779 −2839 −2981 −4004 −3668 −4222 397 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 392(N) −2171 −2655 −1458 −1748 −3334−2364 −2267 −3943 −2365 −3936 −3437 4205 −2932 −2205 −2608 −2224 −2439−3392 −3253 −2909 398 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 393(L) −2871 −2457 −4231 −4103 −1033 −3803 −3165 −541 −37343130 −31 −3935 −3797 −3286 −3484 −3713 −2869 −1136 −2394 −2220 399 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 394(A) 3121−934 −2489 −2561 −3081 −1203 −2295 −2766 −2533 −3080 −2234 −1669 −1953−2234 −2533 936 −746 −1844 −3331 −3090 400 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 395(E) −522 −1773 −240 1676 −2248 −1396−289 −1968 50 −1989 −1115 −174 1198 131 −448 1226 677 −1538 −2214 −1565401 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 396(E)−1481 −3230 1425 2936 −3481 751 −843 −3354 −954 −3256 −2520 −187 −2057−492 −1711 −1193 −1527 −2852 −3445 −2523 402 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 397(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 403 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 398(A) 2847 −932 −2454 −2477 −3066 −1198 −2236 −2763 −2439−3057 −2202 −1635 −1940 −2152 −2471 1777 −731 −1840 −3306 −3056 404 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 399(V) −1771−1603 −3750 −3689 −2037 −3050 −3231 403 −3479 −1154 −1076 −3246 −3399−3383 −3437 −2628 −1917 3536 −3074 −2677 405 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 400(A) 3438 −1472 −2846 −3040 −3287−1726 −2735 −2840 −3028 −3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387−2183 −3405 −3320 406 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 401(K) −2620 −2961 −2461 −2046 −3743 −2791 −1570 −3603 3784−3387 −2839 −2048 −3039 −1260 −465 −2604 −2536 −3331 −3001 −2988 407 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 402(I) −1761−1312 −4317 −3954 −1713 −4027 −3703 3225 −3814 −556 −498 −3712 −3859−3653 −3877 −3344 −1754 2110 −3216 −2787 408 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 403(S) −348 −981 −2200 −2194 −2989 −1227−2073 −2686 −2157 −2970 −2136 −1541 −1946 −1946 −2253 3060 1398 −1824−3217 −2916 409 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 404(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 410 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 405(V) −917−809 −2556 −1976 −827 −2491 −1367 1339 1455 721 94 −1841 −2501 −1487−1710 −1570 −863 2038 −1514 −1151 411 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 406(K) −1386 −2643 −447 1824 −3108 −1893−570 −2762 2860 −2616 −1848 −552 −2117 −166 −3 −1217 −1300 −2388 −2647−2154 412 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *407(N) −537 −1563 −449 −36 −1889 1143 −307 −1529 932 −1655 −844 1794−1658 73 −356 −518 −516 924 −1962 −1392 413 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 408(P) −894 −2181 −369 1705 −2576 −1650−357 −2268 243 −2210 −1375 −330 2093 63 1619 −774 −835 −1876 −2347 −1769414 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 409(V)−419 −634 −1376 −807 1053 −1737 −499 −198 −623 −505 178 600 −1807 −475475 313 −360 1389 −1016 1303 415 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 410(I) −1282 −1082 −3022 −2555 2426 −2683 17672555 −2191 −443 −88 −2038 −2692 −1794 −2075 −1793 −1220 −317 −361 552416 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 411(T)−499 −1595 −431 966 −1830 −1487 −185 −1449 1092 −1574 −754 −207 −1601213 −206 −458 2067 159 −1877 −1296 417 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 412(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 418 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 413(P) −632 −1230 −2074 −2144 −2996 −1453 −2116 −2631 −2128−2928 −2213 −1658 3610 −2006 −2221 −852 1302 −1931 −3185 −2917 419 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 414(A) 3438−1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028 −3257 −2662 −2236 −2447−2798 −2944 −1216 −1387 −2183 −3405 −3320 420 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 415(R) −1454 −2316 −1780 −878−2834 −2232 −428 −2292 2281 −2200 −1473 −940 −2240 −17 2627 −1386 −1270588 −2249 −1960 421 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 416(V) −1771 −1603 −3750 −3689 −2037 −3050 −3231 403 −3479−1154 −1076 −3246 −3399 −3383 −3437 −2628 −1917 3536 −3074 −2677 422 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 417(F) −3342−2776 −4026 −4232 4354 −3545 −1431 −2315 −4038 −1801 −1900 −3299 −3780−3350 −3645 −3490 −3420 −2566 −739 349 423 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 418(D) −1572 −3426 2573 2447 −3613 −1583−879 −3513 −1050 −3393 −2684 1292 −2085 −535 −1855 −1253 −1623 −3000−3585 −2609 424 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 419(S) −879 −1989 1498 −177 −3045 1600 −939 −2843 −904 −2867−2046 −438 −1922 −591 −1483 2171 −1044 −2226 −3072 −2372 425 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 420(E) −2641 −3308 −8963732 −3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560−2479 −2750 −3722 −3563 −3385 426 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 421(Q) −705 −1925 −199 2112 917 −1534 −288−1824 42 −1842 −1054 −210 −1709 2163 −420 −611 −656 −1502 −1997 −1291427 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 422(H)−569 −2048 1450 1526 −2349 −1405 1830 −2103 181 −2058 −1157 −37 −1569272 −349 713 620 −1662 −2240 −1537 428 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 423(C) 1626 2878 −2671 −2107 1264 −1968−1091 233 −1777 −334 250 −1672 −2128 −1459 −1691 −1096 −529 1209 −1066−704 429 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *424(M) −2042 −1634 −4379 −3826 −659 −3976 −2899 2765 −3546 1204 3085−3605 −3604 −2896 −3318 −3183 −1961 195 −2135 −2058 430 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 425(E) 412 −2447 1356 2379−2747 −1477 −445 −2527 −243 −2477 −1622 −107 855 −36 −831 −730 −894−2073 −2668 −1906 431 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 426(A) 2822 −1031 −2418 −2539 −3226 1898 −2364 −2941 −2626−3229 −2379 −1722 −2026 −2302 −2634 −654 −848 −1983 −3415 −3226 432 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 427(I) −1772−1325 −4307 −3877 −1405 −3993 −3383 2935 −3705 820 −217 −3632 −3761−3400 −3682 −3260 −1742 2033 −2838 −2525 433 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 428(L) −875 −1634 −575 959 −1581 −1769−525 −1179 −135 1884 −625 −547 −1931 1405 −450 −909 −816 −1074 −1883−1383 434 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *429(A) 1705 −1826 −180 949 −2318 −1410 −359 −2041 −53 −2067 −1204 1001−1652 52 −561 1232 −595 −1609 −2298 −1643 435 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 430(D) −1074 −2458 2381 60 −29211927 −658 −2710 −463 −2675 −1860 −271 −1918 −276 866 −915 −1100 −2245−2845 −2124 436 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 431(K) −688 −2117 785 888 −2469 −1529 −187 −2189 2380 −2106−1221 −162 −1661 256 1134 −553 −619 −1760 −2240 −1607 437 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 432(I) −2019 −1582−4380 −3941 −1000 −4086 −3253 3295 −3671 1100 145 −3736 −3783 −3222−3556 −3378 −1976 657 −2517 −2289 438 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 433(Q) −490 −1797 −369 171 −2078 −14571762 −1779 1157 −1780 −905 1165 −1550 1798 −48 −396 −422 725 −1986 −1366439 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 434(A)1954 −1836 1733 −180 −2714 −1429 −806 −2438 −679 −2518 −1698 −430 1775−448 −1211 −736 −894 −1923 −2765 −2117 440 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 435(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 441 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 436(D) −1736 −3455 3490 97 −3737 −1646 −1070 −3753 −1363 −3647−3016 1602 −2204 −760 −2218 −1420 −1838 −3213 −3756 −2780 442 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 437(V) −1721 −1302−4229 −3874 −1705 −3894 −3582 1607 −3706 −582 −513 −3610 −3786 −3559−3767 −3209 −1725 3294 −3158 −2712 443 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 438(V) 594 −988 −3391 −2911 −1164 −2888−2187 845 −2637 765 −154 −2576 −2962 −2387 −2622 −2074 −1205 2800 −2084−1724 444 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *439(V) −1771 −1603 −3750 −3689 −2037 −3050 −3231 403 −3479 −1154 −1076−3246 −3399 −3383 −3437 −2628 −1917 3536 −3074 −2677 445 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 440(I) −1754 −1308 −4295−3867 −1434 −3978 −3377 2661 −3697 862 −247 −3617 −3754 −3406 −3679−3243 −1725 2373 −2852 −2526 446 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 441(R) −2957 −3022 −3318 −2735 −3796 −2998−1968 −3912 −846 −3631 −3157 −2611 −3280 −1724 4056 −3026 −2913 −3650−3096 −3185 447 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 442(Y) −1321 −1438 −1994 −1608 2186 527 −450 −1117 −1481 −1211−693 1178 −2522 −1217 −1665 −1518 −1275 −1021 −198 3178 448 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 443(C) −675 2205 −2544972 −572 −2236 −1121 1373 −1671 679 261 −1700 −2270 −1403 −1668 −1311−621 1601 −1150 −790 449 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 444(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761−3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222450 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 445(P)−2931 −2878 −3420 −3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165 −34914225 −3781 −3695 −3182 −3279 −4087 −3594 −4064 451 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 446(K) −1060 −2058 −1088 −460−2432 −1917 −357 −1970 2801 −1978 −1220 −632 −1990 1339 367 −999 −946536 −2145 −1717 452 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 447(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 453 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 448(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 454 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 449(P) −2931 −2878 −3420 −3706−4181 −2925 −3468 −4621 −3859 −4490 −4165 −3491 4225 −3781 −3695 −3182−3279 −4087 −3594 −4064 455 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 450(G) −2594 −2690 −3304 −3623 −4328 3747 −3462−4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668−4222 456 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *451(M) −2406 −2296 −3638 −3594 −1525 −3105 −2824 −1047 −3121 −596 5043−3293 −3425 −3046 −2996 −2911 −2552 −1398 −2513 −2207 457 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 452(P) −1659 −2241−2022 −1646 −3185 −2242 −1373 −3000 −450 −2936 −2274 −1624 3435 −10652095 −1730 −1750 −2593 −2816 −2613 458 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 453(E) −2641 −3308 −896 3732 −3966 −2458−2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560 −2479 −2750 −3722−3563 −3385 459 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 454(M) −2406 −2296 −3638 −3594 −1525 −3105 −2824 −1047 −3121−596 5043 −3293 −3425 −3046 −2996 −2911 −2552 −1398 −2513 −2207 460 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 455(L) −2871−2457 −4231 −4103 −1033 −3803 −3165 −541 −3734 3130 −31 −3935 −3797−3286 −3484 −3713 −2869 −1136 −2394 −2220 461 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 456(K) 1368 −1491 −763 −332 −2319−1417 −551 −1998 1786 −2068 −1221 −500 −1721 −160 −470 1631 −587 −1532−2299 −1754 462 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 457(P) −1500 −1738 −2514 −2380 −1555 −2358 −2022 −1126 −20631224 −841 −2189 3436 −2061 −2129 −1822 −1674 −1231 −2290 −1878 463 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 458(T) −351−974 −2208 −2185 −2894 −1237 −2041 −2561 −2125 −2863 −2046 −1539 −1948−1923 −2218 1543 3230 −1758 −3139 −2834 464 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 459(S) −897 −1462 −2333 −2543 −3185−1640 −2474 −3294 −2686 −3497 −2780 −1973 −2360 −2483 −2703 3465 −1316−2413 −3310 −3025 465 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 460(M) 2706 −986 −2433 −2144 −1502 −1684 −1706 −700 −1858 −9682744 −1705 −2188 −1713 −1932 −963 −862 −592 −2145 −1794 466 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 461(I) −2103 −1659−4461 −3992 −869 −4152 −3233 3082 −3723 1619 290 −3801 −3788 −3171 −3557−3432 −2046 487 −2418 −2265 467 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 462(I) −1761 −1312 −4317 −3954 −1713 −4027 −37033225 −3814 −556 −498 −3712 −3859 −3653 −3877 −3344 −1754 2110 −3216−2787 468 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *463(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212−3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 469 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 464(K) 1641 −2033 −323914 −2415 −1565 −296 −2097 2052 −2080 −1233 −257 −1736 125 −133 −646−702 −1707 −2258 −1657 470 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 465(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761−3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222471 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 466(L)−1699 −1807 −2268 −1925 −830 −2795 −1551 −455 −1225 2510 90 −1958 −28451927 −1308 −2067 −1651 −846 −1841 −1454 472 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 467(G) −2594 −2690 −3304 −3623 −43283747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981−4004 −3668 −4222 473 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 468(D) −853 −2415 2115 1717 −2702 −1468 −378 −2484 1085 −2417−1546 −84 −1732 41 −699 696 −824 −2025 −2594 −1839 474 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 469(S) −892 −1780 −931 −688−2757 −1643 −830 −2472 1671 −2492 −1708 −799 −2018 −468 −365 2676 −1004−1981 −2598 −2130 475 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 470(C) −1135 3503 −3700 −3406 −1670 −2549 −2675 653 −3101 −916−667 −2727 −2925 −2870 −3030 −1868 −1288 2927 −2619 −2222 476 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 471(A) 2590 −1035−2404 −2530 −3236 2290 −2365 −2954 −2627 −3240 −2389 −1719 −2027 −2302−2637 −656 −851 −1991 −3423 −3234 477 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 472(L) −2632 −2152 −4630 −4185 1767−4324 −2442 −61 −3879 2789 563 −3833 −3823 −2970 −3513 −3609 −2518 −738−1527 −945 478 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 473(I) −2073 −1632 −4434 −3975 −911 −4130 −3238 3164 −37061451 244 −3779 −3785 −3187 −3557 −3413 −2021 546 −2449 −2273 479 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 474(T) −1213 −1674−2755 −2906 −3163 −1922 −2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708−2753 −1463 3819 −2197 −3286 −3156 480 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 475(D) −2784 −3432 4016 −1200 −4140−2466 −2197 −4505 −2621 −4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938−4046 −3710 −3552 481 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 476(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 482 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 477(R) −2957−3022 −3318 −2735 −3796 −2998 −1968 −3912 −846 −3631 −3157 −2611 −3280−1724 4056 −3026 −2913 −3650 −3096 −3185 483 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 478(F) −3342 −2776 −4026 −4232 4354−3545 −1431 −2315 −4038 −1801 −1900 −3299 −3780 −3350 −3645 −3490 −3420−2566 −739 349 484 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 479(S) −897 −1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686−3497 −2780 −1973 −2360 −2483 −2703 3465 −1316 −2413 −3310 −3025 485 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 480(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 486 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 481(G) −2594 −2690 −3304 −3623−4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839−2981 −4004 −3668 −4222 487 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 482(T) −359 −976 −2225 −2229 −2900 −1242 −2074−2560 −2170 −2875 −2064 −1561 −1958 −1969 −2247 1110 3375 −1760 −3152−2850 488 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *483(Y) −3402 −2632 −3941 −4011 1064 −3924 3388 −2526 −3541 −1996 −1973−2625 −3821 −2664 −3170 −3135 −3280 −2619 3420 3756 489 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 484(G) −2594 −2690 −3304−3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779−2839 −2981 −4004 −3668 −4222 490 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 485(M) −2322 −1904 −4536 −3951 2387 −4112−2676 67 −3649 2034 3156 −3710 −3633 −2803 −3311 −3309 −2204 −588 −1794−1586 491 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *486(V) −1771 −1603 −3750 −3689 −2037 −3050 −3231 403 −3479 −1154 −1076−3246 −3399 −3383 −3437 −2628 −1917 3536 −3074 −2677 492 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 487(V) −1771 −1603 −3750−3689 −2037 −3050 −3231 403 −3479 −1154 −1076 −3246 −3399 −3383 −3437−2628 −1917 3536 −3074 −2677 493 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 488(G) −2594 −2690 −3304 −3623 −4328 3747−3462 −4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004−3668 −4222 494 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 489(H) −3205 −3079 −2723 −2890 −2110 −3046 5295 −4135 −2617−3813 −3561 −2886 −3482 −2833 −2620 −3291 −3356 −3895 −2397 −1681 495 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 490(V) −1754−1297 −4329 −3968 −1770 −4053 −3752 2604 −3840 −621 −545 −3728 −3878−3699 −3917 −3370 −1746 2859 −3276 −2829 496 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 491(A) 2587 −828 −2477 −2155 −1837 −1468−1728 −743 −1941 −1564 −954 −1607 −2033 −1725 −2034 −738 1178 1108 −2310−1972 497 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *492(P) −2931 −2878 −3420 −3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165−3491 4225 −3781 −3695 −3182 −3279 −4087 −3594 −4064 498 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 493(E) −2641 −3308 −896 3732−3966 −2458 −2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560 −2479−2750 −3722 −3563 −3385 499 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 494(A) 3438 −1472 −2846 −3040 −3287 −1726 −2735−2840 −3028 −3257 −2662 −2236 −2447 −2798 −2944 −1216 −1387 −2183 −3405−3320 500 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *495(Y) −866 −976 −1863 −1331 1353 −2145 1318 −556 −1116 −777 −173 −1242−2197 1714 −1301 −1173 −802 888 −445 2749 501 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 496(D) 417 −1831 1647 1094 −2065−1488 −353 −1618 −107 −1820 −1019 −189 −1698 30 −623 −603 −643 1629−2154 −1520 502 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 497(G) −2594 −2690 −3304 −3623 −4328 3747 −3462 −4761 −3953−4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668 −4222 503 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 498(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 504 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 499(T) 492 −1190 −706 −181 −1475311 −333 −1099 −81 71 −509 570 1113 −6 −509 −450 1123 −835 −1680 −1161505 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 500(I)−2091 −1746 −3971 −3840 −1676 −3532 −3289 3684 −3581 −659 −693 −3562−3674 −3445 −3521 −3194 −2146 449 −2877 −2493 506 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 501(A) 3103 −1036 −2445 −2572−3222 1051 −2380 −2930 −2650 −3226 −2381 −1739 −2034 −2327 −2648 −664−857 −1981 −3412 −3228 507 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 502(L) −2239 −1892 −3711 −3400 301 −3520 −1210 −542 −29482564 −35 −2786 −3395 −2438 −2750 −2747 −2165 −945 −573 2562 508 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 503(V) −1757 −1387−4101 −3681 −1174 −3714 −3031 880 −3410 1254 −60 −3407 −3585 −3094 −3354−2984 −1743 3014 −2536 −2219 509 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 504(Q) −982 −2251 −866 971 −2711 −1822 −252−2340 1444 −2194 −1356 −464 −1885 2646 1632 −858 −863 −1958 −2245 −1765510 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 505(E)−1162 −2771 2137 2239 −3046 −1526 −626 −2849 −546 −2792 −1983 −145 −1905−242 −1192 −940 1396 −2385 −2990 −2169 511 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 506(G) −1707 −2684 1591 −614 −3783 3190−1613 −3795 −1887 −3775 −3119 −915 −2456 −1358 −2539 −1610 −1924 −3150−3636 −3124 512 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 507(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621−4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 513 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 508(M) −473−522 −1819 −1236 −468 −1879 −687 1519 −996 566 1677 −1154 −1937 836−1131 1079 −413 102 −957 −585 514 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 509(I) −1761 −1312 −4317 −3954 −1713 −4027−3703 3225 −3814 −556 −498 −3712 −3859 −3653 −3877 −3344 −1754 2110−3216 −2787 515 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 510(T) 782 −1467 −550 1029 −2202 −1425 −709 −1791 −472 −1993−1203 −528 −1787 −368 −902 −617 2685 −1400 −2333 −1783 516 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 511(I) −1766 −1333−4283 −3923 −1635 −3967 −3619 3388 −3759 −473 −437 −3672 −3822 −3576−3804 −3285 −1764 1695 −3126 −2717 517 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 512(D) −2784 −3432 4016 −1200 −4140−2466 −2197 −4505 −2621 −4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938−4046 −3710 −3552 518 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 513(A) 2705 −1451 −1036 −913 −2506 −1504 −1143 −2174 −794−2337 −1613 −946 −1993 2040 −1061 −809 −910 −1703 −2633 −2156 519 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 514(H) −615 −16801444 66 −1883 168 2650 −1558 −86 −1691 −891 −223 −1680 31 −577 −571 −5851267 −2007 −1397 520 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 515(K) −654 −2006 −546 42 −2376 −1581 −133 −2066 1935 −1987−1107 1132 −1658 1043 1058 −540 1180 −1660 −2113 −1532 521 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 516(N) −933 −2085 −946−284 −2472 −1822 −253 −2090 1711 76 −1204 1918 −1876 175 1799 −841 −817−1755 −2132 −1663 522 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 517(E) −416 −987 −843 1107 −1070 −1583 −338 −623 −183 879 −172−489 −1679 −94 −565 544 813 265 −1379 −905 523 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 518(I) −2258 −1804 −4588 −4084−706 −4269 −3231 2527 −3807 2292 465 −3923 −3814 −3118 −3570 −3544 −2181190 −2303 −2237 524 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 519(Q) −477 −1909 958 282 −2211 −1389 1484 −1953 285 −1921−1018 −32 −1517 2318 −225 630 559 −1525 −2110 −1430 525 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 520(L) −2127 −1743 −4402−3796 1257 −3918 −2674 149 −3492 2527 2164 −3553 −3509 −2714 −3181 −3095−2019 570 −1870 −1818 526 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 521(N) −723 −2217 958 236 −2518 −1466 1611 −2279 1719−2217 −1334 2285 −1666 166 −401 −570 −677 −1837 −2382 −1678 527 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 522(V) −1754 −1297−4330 −3968 −1770 −4053 −3752 2623 −3841 −620 −545 −3729 −3878 −3699−3918 −3371 −1746 2846 −3277 −2830 528 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 523(S) 1545 −974 −2003 −1825 −2867 −1206−1790 −2580 −1788 −2795 −1932 −1362 1826 −1586 −1999 2362 −672 −1755−3057 −2721 529 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 524(D) −1776 −3649 3326 1869 −3838 −1642 −1031 −3788 −1322−3660 −3029 −245 −2192 −711 −2201 −1425 −1855 −3264 −3821 −2816 530 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 525(E) 423−2950 1944 2696 −3223 −1545 −718 −3047 −715 −2979 −2196 −161 −1968 −347−1403 −1043 −1314 −2569 −3177 −2316 531 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 526(E) −2641 −3308 −896 3732 −3966 −2458−2043 −4105 −2128 −4016 −3555 −1531 −2959 −1842 −2560 −2479 −2750 −3722−3563 −3385 532 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 527(L) −2339 −1899 −4618 −4042 1570 −4204 −2849 1440 −37582558 676 −3825 −3700 −2902 −3418 −3418 −2226 −382 −1924 −1778 533 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 528(A) 2338 −1990−241 938 −2395 −1557 −423 −2061 954 −2103 −1286 −301 −1791 −26 −375 −717−784 −1691 −2330 −1728 534 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 529(R) 524 −2098 −789 −146 −2504 −1729 1632 −2153 1229−2054 −1204 −379 −1789 1328 2313 −719 −724 −1774 −2150 −1637 535 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369−294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 530(R) −2957 −3022−3318 −2735 −3796 −2998 −1968 −3912 −846 −3631 −3157 −2611 −3280 −17244056 −3026 −2913 −3650 −3096 −3185 536 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 531(R) −1895 −2713 −2327 −1192 −3484−2502 −481 −2856 2144 −2544 −1842 −1161 −2458 1393 3023 −1770 −1619−2599 −2421 −2259 537 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 532(A) 2935 −1714 −553 857 −2769 −1546 −1218 −2333 −1106 −2591−1873 −809 −2065 −934 −1502 −954 −1103 −1872 −2898 −2374 538 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 533(A) 1291 −1874 −1761227 −2177 −1392 −109 −1909 277 −1891 −995 1134 −1522 1248 −228 −361 562−1492 −2090 −1419 539 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 534(W) −805 −687 −2581 −2028 138 −2236 −697 897 −1681 −421 141−1645 −2282 −1369 −1627 −1315 636 −90 4479 1809 540 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 —−16 −7108 −8150 −894 −1115 −701 −1378 * * 535(H) −408 −1801 −274 1284−2096 −1385 1500 −1822 1168 −1802 −899 −33 −1479 1381 −102 −303 595 221−1996 −1339 541 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 536(Q) −650 −1737 −627 −72 −1981 −1615 −209 −1625 1223 392−866 −318 1222 2120 50 −598 −572 −1326 −1932 −1394 542 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 537(P) −2931 −2878 −3420−3706 −4181 −2925 −3468 −4621 −3859 −4490 −4165 −3491 4225 −3781 −3695−3182 −3279 −4087 −3594 −4064 543 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −324 −7108 −2368−894 −1115 −701 −1378 * * 538(A) 2195 −924 −968 −546 −1397 −1356 −583−812 −365 −1167 −487 −618 −1660 1324 −684 −483 −404 462 −1703 −1242 544— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −19 −6804 −7846 −894 −1115 −428 −1961 * * 539(P) 411−1017 −1886 −1616 −1600 −1588 −1411 −962 −1408 495 −755 −1384 3156 −1323−1577 −847 −785 −783 −2111 −1716 545 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 540(R) −1612 −2397 −2037 −1033 −2897−2352 −458 −2365 2184 665 −1520 −1051 −2334 −51 2602 −1545 −1395 −2143−2262 −2014 546 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 541(Y) 712 −796 −2334 −1883 −370 −2028 −986 −143 −1607 −663−131 −1587 −2243 −1383 −1656 −1178 −771 1114 −965 3479 547 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294−249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 542(T) −527 −1669 1091−27 −2315 −1379 −443 −2033 −151 −2081 −1218 −282 557 −41 −650 1128 2077−1576 −2321 −1690 548 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 543(R) −2957 −3022 −3318 −2735 −3796 −2998 −1968 −3912 −846−3631 −3157 −2611 −3280 −1724 4056 −3026 −2913 −3650 −3096 −3185 549 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 544(G) −2594−2690 −3304 −3623 −4328 3747 −3462 −4761 −3953 −4671 −4212 −3320 −3352−3748 −3779 −2839 −2981 −4004 −3668 −4222 550 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 545(V) −1747 −1296 −4310 −3948−1758 −4023 −3716 2215 −3813 −615 −540 −3705 −3860 −3670 −3887 −3339−1741 3087 −3252 −2806 551 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 546(L) −2871 −2457 −4231 −4103 −1033 −3803 −3165 −541−3734 3130 −31 −3935 −3797 −3286 −3484 −3713 −2869 −1136 −2394 −2220 552— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 547(A) 2404−890 −1926 −1629 −1803 1275 −1415 −1282 −1490 392 −963 −1316 −1930 −1328−1674 −654 −644 −952 −2187 −1810 553 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 548(K) −2620 −2961 −2461 −2046 −3743−2791 −1570 −3603 3784 −3387 −2839 −2048 −3039 −1260 −465 −2604 −2536−3331 −3001 −2988 554 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 549(Y) −3621 −2707 −4176 −4424 2950 −4049 −394 −2539 −4002−1942 −1987 −2749 −3933 −2854 −3451 −3299 −3499 −2690 349 4094 555 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 550(A) 3438−1472 −2846 −3040 −3287 −1726 −2735 −2840 −3028 −3257 −2662 −2236 −2447−2798 −2944 −1216 −1387 −2183 −3405 −3320 556 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16−7108 −8150 −894 −1115 −701 −1378 * * 551(H) −1741 −2627 −2070 −1046−3303 −2401 2713 −2751 2478 −2476 −1755 −1061 −2375 −27 2379 −1621 −1497−2477 −2379 −2161 557 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 552(L) −1014 −876 −2956 −2408 −582 −2550 −1529 1721 −2079 2042345 −2114 −2581 −1775 −2028 454 −980 286 −1414 −1096 558 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 553(V) 933 −842 −2818 −2467−1542 −1870 −1890 154 −2226 −1095 −617 −1932 −2326 −1995 −2259 −11261070 2769 −2180 −1826 559 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115−701 −1378 * * 554(S) −787 −1522 −1486 −1172 −2714 −1599 −1112 −2500−433 −2563 −1791 −1110 −2067 −796 1351 2916 −989 −1943 −2648 −2234 560 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 555(S) −326−1010 −1779 −1541 −2691 −1234 −1566 −2386 −1486 −2594 −1749 −1228 1196−1330 −1747 2396 1967 −1662 −2876 −2496 561 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108−8150 −894 −1115 −701 −1378 * * 556(A) 3121 −934 −2489 −2561 −3081 −1203−2295 −2766 −2533 −3080 −2234 −1669 −1953 −2234 −2533 936 −746 −1844−3331 −3090 562 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 557(S) −897 −1462 −2333 −2543 −3185 −1640 −2474 −3294 −2686−3497 −2780 −1973 −2360 −2483 −2703 3465 −1316 −2413 −3310 −3025 563 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * * 558(R) −586−1873 −516 979 −2188 −1543 −123 −1869 1290 −353 −980 −202 −1622 314 1886−491 782 −1495 −2024 −1439 564 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894−1115 −701 −1378 * * 559(G) −2594 −2690 −3304 −3623 −4328 3747 −3462−4761 −3953 −4671 −4212 −3320 −3352 −3748 −3779 −2839 −2981 −4004 −3668−4222 565 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 4596 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701 −1378 * *560(C) 2804 3772 −3185 −3198 −2739 −1303 −2462 −2065 −2882 −2628 −1924−1927 −2044 −2547 −2727 −661 −799 −1463 −3099 −2886 566 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249— −16 −7108 −8150 −894 −1115 −701 −1378 * * 561(V) −1771 −1603 −3750−3689 −2037 −3050 −3231 403 −3479 −1154 −1076 −3246 −3399 −3383 −3437−2628 −1917 3536 −3074 −2677 567 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150−894 −1115 −701 −1378 * * 562(T) −1213 −1674 −2755 −2906 −3163 −1922−2659 −2698 −2788 −3105 −2612 −2311 −2600 −2708 −2753 −1463 3819 −2197−3286 −3156 568 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275394 45 96 359 117 −369 −294 −249 — −16 −7108 −8150 −894 −1115 −701−1378 * * 563(D) −2784 −3432 4016 −1200 −4140 −2466 −2197 −4505 −2621−4365 −3956 −1551 −3014 −2039 −3232 −2593 −2938 −4046 −3710 −3552 569 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117−369 −294 −249 — −21 −6715 −7757 −894 −1115 −701 −1378 * * 564(F) −525−445 −2202 −1627 1946 −2001 −744 1247 −1346 952 561 1079 −2030 −1067−1362 −1067 −465 338 −714 −230 570— * * * * * * * * * * * * * * * * * * * * — * * * * * * * * 0

What claimed is:
 1. A method of making isobutanol comprising: (a)providing a recombinant yeast host cell comprising: (i)at least oneheterologous polynucleotide encoding a dihydroxy-acid dehydrataseenzyme, wherein the dihydroxy-acid dehydratase enzyme has an amino acidsequence with at least 95% identity to SEQ ID NO: 958, and (ii) at leastone deletion in an endogenous gene encoding a polypeptide affecting Fe—Scluster biosynthesis, wherein the gene encoding the polypeptideaffecting Fe—S cluster biosynthesis is selected from Fe Repressor ofActivation-2 (FRA2), glutaredoxin-3 (GRX3), and CCC1, wherein therecombinant yeast host cell produces isobutanol; and (b) contacting therecombinant yeast host cell of (a) with a fermentable carbon substratein a fermentation medium under conditions wherein isobutanol isproduced.
 2. The method of claim 1, wherein the polypeptide affectingFe-S cluster biosynthesis is FRA2.
 3. The method of claim 1, wherein therecombinant yeast host cell is selected from the group consisting ofSaccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces,Yarrowia, Issatchenkia, and Pichia.
 4. The method of claim 1, whereinthe dihydroxy-acid dehydratase enzyme is expressed in the cytosol of therecombinant yeast host cell.
 5. The method of claim 1, wherein therecombinant yeast host cell comprises an isobutanol biosyntheticpathway.
 6. The method of claim 1, wherein the isobutanol is isolatedfrom the fermentation medium by distillation, azeotropic distillation,liquid-liquid extraction, adsorption, gas stripping, membraneevaporation, or pervaporation.
 7. The method of claim 1, wherein solidsare removed from the fermentation medium by centrifugation, filtration,or decantation.